基于Sirt1/FoxO1通路探讨瑞马唑仑减轻脓毒症小鼠脑损伤的机制研究

Mechanism of remazolam in reducing brain injury in sepsis mice based on Sirt1/FoxO1 pathway

目的探究瑞马唑仑对脓毒症引起的脑损伤的影响及机制。方法雄性C57BL/6J小鼠,采用随机数字表法分为假手术组、模型组、瑞马唑仑组(8 mg/kg)、瑞马唑仑+Sirt1抑制剂(EX527)组(8 mg/kg瑞马唑仑+5 mg/kg EX527)、EX527组,每组38只。采用盲肠结扎穿孔法(CLP)制备脓毒症相关性脑病(SAE)小鼠模型。给予相应的干预后,观察并记录术后7 d内小鼠存活率,采用Morris水迷宫检测小鼠逃避潜伏期和穿越平台次数;手术后24 h,通过伊文斯蓝(EB)渗漏量评估血脑屏障(BBB)通透性,酶联免疫吸附测定(ELISA)法检测脑组织白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α、IL-1β水平,化学比色法检测脑组织丙二醛(MDA)水平和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性,HE染色观察海马组织病理学变化,TUNEL法检测神经元凋亡,蛋白免疫印迹(Western Blot)检测海马组织沉默信息调节因子1(Sirt1)/叉头框蛋白O1(FoxO1)通路相关蛋白表达。结果与假手术组相比,模型组小鼠的存活率、穿越平台次数、SOD和CAT活性、Sirt1和胞质NF-κB p65蛋白水平显著下降,逃避潜伏期、脑组织EB含量、IL-6、TNF-α、IL-1β和MDA水平、海马神经元凋亡指数、乙酰化FoxO1(Ac-FoxO1)/FoxO1比值和乙酰化核因子-κB p65(Ac-NF-κB p65)、胞核NF-κB p65蛋白水平显著升高(均P<0.05);与模型组相比,瑞马唑仑组小鼠的存活率、穿越平台次数、SOD和CAT活性、Sirt1和胞质NF-κB p65蛋白水平显著升高,逃避潜伏期、脑组织EB含量、IL-6、TNF-α、IL-1β和MDA水平、海马神经元凋亡指数、Ac-FoxO1/FoxO1比值和Ac-NF-κB p65、胞核NF-κB p65蛋白水平显著降低(均P<0.05);EX527可抑制Sirt1表达,显著减弱瑞马唑仑对SAE小鼠的上述保护作用(均P<0.05)。结论瑞马唑仑可提高脓毒症小鼠存活率,通过维持BBB完整性、抑制神经炎症和氧化应激,减少神经元凋亡,减轻脓毒症引起的脑损伤;其作用机制可能与激活Sirt1/FoxO1通路,抑制NF-κB活化有关。

Objective To explore the effect and action of remazolam on brain injury caused by sepsis.Methods Male C57BL/6J mice were randomly separated into sham operation,model,remazolam(8 mg/kg),remazolam+Sirt1 inhibitor(EX527)(8 mg/kg remazolam+5 mg/kg EX527),and EX527 groups by the random number table method,with 38 rats in each group.A sepsis⁃associated encephalopathy(SAE)mouse model was established by cecal ligation and puncture.After the corresponding intervention was given,the survival rates of mice within seven days after the operation were observed and recorded,and a Morris water maze was used to detect the mice’s escape latency and number of times crossing the platform.Twenty⁃four hours after surgery,blood⁃brain barrier(BBB)permeability was assessed by Evans blue(EB)leakage;the levels of interleukin(IL)⁃6,tumor necrosis factor(TNF)⁃α,and IL⁃1βin brain tissue were detected by enzyme⁃linked immunosorbent assay;the levels of malondialdehyde(MDA)and activities of superoxide dismutase(SOD)and catalase(CAT)in brain tissue were detected by chemical colorimetry.Histopathological changes to the hippocampus were observed by HE staining,and neuronal apoptosis was detected using the TUNEL method.Western Blot was used to detect the expression of silent information regulator 1(Sirt1)/forkhead box O1(FoxO1)pathway⁃related proteins in the hippocampus.Results Compared with the sham group,mice in the model group had a significantly decreased survival rate,platform crossing time,SOD and CAT activities,and Sirt1 and cytoplasmic NF⁃κB p65 protein levels and significantly increased brain tissue EB content;IL⁃6,TNF⁃α,IL⁃1β,and MDA levels;hippocampal neuron apoptosis index;acetylated FoxO1(Ac⁃FoxO1)/FoxO1 ratio;and acetylated nuclear factor⁃κB p65(Ac⁃NF⁃κB p65)and nuclear NF⁃κB p65 protein levels(all P<0.05).Compared with the model group,mice in the remazolam group had a significantly increased survival rate,platform crossing time,SOD and CAT activities,and Sirt1 and cytoplasmic NF⁃κB p65 protein levels,and significantly decreased escape latency;brain tissue EB content;IL⁃6,TNF⁃α,IL⁃1β,and MDA levels;hippocampal neuron apoptosis index;Ac⁃FoxO1/FoxO1 ratio;and Ac⁃NF⁃κB p65 and nuclear NF⁃κB p65 protein levels(all P<0.05).EX527 inhibited the expression of Sirt1 and substantially attenuated the above protective effects of remazolam in SAE mice(all P<0.05).Conclusions Remazolam improved the survival rate of mice with sepsis and reduced neuronal apoptosis and brain damage caused by sepsis by maintaining BBB integrity and inhibiting neuroinflammation and oxidative stress.Its mechanism of action may be related to the activation of the Sirt1/FoxO1 pathway and inhibition of NF⁃κB activation.