虾青素预处理通过调控Sirt1/miR-134信号通路改善脑缺血再灌注大鼠认知功能

Astaxanthin preconditioning ameliorates cognitive function in rats with cerebral ischemia/reperfusion by regulating Sirt1/miR-134 signaling pathway

目的:探究虾青素(ATX)预处理是否通过调控沉默信息调节因子1(Sirt1)/微小RNA-134(miR-134)信号通路影响脑缺血再灌注(CI/R)大鼠认知功能。方法:将60只雄性SD大鼠随机分为假手术组(sham组)、模型组(CI/R组)、ATX低剂量(50 mg/kg)组(ATX-L组)、ATX高剂量(100 mg/kg)组(ATX-H组)和ATX(100 mg/kg)+Sirt1抑制剂EX527(10 mg/kg)组(ATX+EX527组),每组12只。分组完成后给予相应的药物进行预处理,每天1次,连续3 d。末次给药1 h后,建立CI/R大鼠模型。再灌注24 h后对大鼠进行神经功能缺损评分,并采用Morris水迷宫实验检测大鼠的认知功能;HE染色观察海马神经元损伤;TUNEL染色检测海马神经元凋亡;免疫组化法检测海马组织脑源性神经营养因子(BDNF)的表达;RT-qPCR检测海马组织miR-134水平及Sirt1、环磷酸腺苷反应元件结合蛋白(CREB)和BDNF mRNA表达;Western blot检测海马组织Sirt1、CREB和BDNF蛋白表达。结果:与sham组相比,CI/R组大鼠神经功能缺损评分、海马神经元凋亡率、miR-134水平和逃避潜伏期均增加,空间探索实验中在目标象限停留时间和穿越平台次数、Sirt1和BDNF的mRNA和蛋白表达、CREB的mRNA表达及p-CREB/CREB比值降低(P<0.05),海马神经元数量减少;与CI/R组相比,ATX-L组和ATX-H组大鼠神经功能缺损评分、海马神经元凋亡率、miR-134水平和逃避潜伏期均降低(P<0.05),在目标象限停留时间和穿越平台次数、Sirt1和BDNF的mRNA和蛋白表达、CREB的mRNA表达及p-CREB/CREB比值升高(P<0.05),海马神经元数量增加;在ATX干预的基础上使用EX527可明显上调miR-134的表达,减弱ATX对CI/R后大鼠认知功能的改善作用和对CREB/BDNF信号通路的激活作用。结论:ATX预处理可能通过调控Sirt1/miR-134信号通路,激活CREB/BDNF信号通路,进而改善CI/R后大鼠的认知功能。

AIM:To investigate the effect of astaxanthin(ATX)preconditioning on cognitive function in rats with cerebral ischemia/reperfusion(CI/R),to explore the role of silent information regulator 1(Sirt1)/microRNA-134(miR-134)signaling pathway in this process.METHODS:A total of 60 male SD rats were randomly divided into sham operation group(sham group),model group(CI/R group),low-dose(50 mg/kg)ATX group(ATX-L group),high-dose(100 mg/kg)ATX group(ATX-H group)and ATX(100 mg/kg)+Sirt1 inhibitor EX527(10 mg/kg)group(ATX+EX527 group),with 12 rats in each group.After grouping,the corresponding drugs were given for pretreatment,once a day for 3 consecutive days.One hour after the last administration,a CI/R rat model was established.After 24 h of reperfusion,the rats were scored for neurological deficits,and the Morris water maze test was used to detect the cognitive function of the rats.HE staining was used to observe hippocampal neuron damage.TUNEL staining was used to detect the apoptosis of hippocampal neurons.Immunohistochemical method was used detect the expression of brain-derived neurotrophic factor(BDNF)in the hippocampus.RT-qPCR and Western blot were used to detect the expression of miR-134,Sirt1,cAMP response element binding protein(CREB)and BDNF.RESULTS:Compared with sham group,the neurological deficit score,hippocampal neuron apoptotic rate,miR-134 level and escape latency were increased in CI/R group(P<0.05),while the staying time in the target quadrant and the number of crossing the platform in the space exploration experiment,the mRNA and protein expression of Sirt1 and BDNF,the mRNA expression of CREB,the p-CREB/CREB ratio,and the number of hippocampal neurons were decreased(P<0.05).Compared with CI/R group,the neurological deficit score,hippocampal neuron apoptotic rate,miR-134 level and the escape latency were decreased in ATX-L group and ATX-H group(P<0.05),while the staying time in the target quadrant and the number of crossing the platform in the space exploration experiment,the mRNA and protein expression of Sirt1 and BDNF,the mRNA expression of CREB,the p-CREB/CREB ratio,and the number of hippocampal neurons were increased(P<0.05).EX527 significantly up-regulated the expression of miR-134 on the basis of ATX intervention,and weakened the improvement effect of ATX on cognitive function of the rats after CI/R and the activation effect of ATX on CREB/BDNF signaling pathway.CONCLUSION:Pretreatment with ATX may activate the CREB/BDNF signaling pathway by regulating the Sirt1/miR-134 signaling pathway,thereby improving the cognitive function of rats after CI/R.