摘要
目的 评价腺苷酸活化蛋白激酶(AMPK)-沉默信息调节因子 1(SIRT1)-核因子-κB(NF-κB)信号通路在三七总皂苷(PNS)减轻机械通气大鼠脑损伤中的作用.方法 SPF级雄性SD大鼠72只,10周龄,体质量357～377 g,采用随机数字表法将大鼠分为6组(n=12):假手术组、模型组、PNS低剂量组、PNS中剂量组、PNS高剂量组和PNS高剂量+compound C组.PNS低剂量组、PNS中剂量组和PNS高剂量组分别腹腔注射PNS 12.5、25.0和50.0 mg/kg;PNS高剂量+compound C组在腹腔注射PNS 50 mg/kg后10 min时,尾静脉注射compound C 0.2 mg/kg;假手术组和模型组腹腔注射10 ml/kg生理盐水.各组于机械通气前30 min注射药物或生理盐水.机械通气模型:模型组通气频率40次/min,潮气量40 ml/kg,机械通气6 h;假手术组机械通气6 h,潮气量10 ml/kg.采用Morris水迷宫实验检测大鼠学习记忆功能,采用ELISA法检测血清IL-1β、IL-6、TNF-α及多巴胺(DA)浓度,尼氏染色进行海马CA1区神经元计数,采用Western blot法检测海马CA1区P2Y1嘌呤受体(P2Y1R)、(肌)营养不良短小蛋白结合蛋白1(dysbindin-1)、AMPK的表达、磷酸化SIRT1(p-SIRT1)/SIRT1比值和磷酸化NF-κB p65(p-NF-κB p65)/NF-κB比值.结果 与假手术组比较,模型组逃避潜伏期延长,穿越原平台位置次数减少,目标象限停留时间缩短,血清IL-1β、IL-6和TNF-α浓度升高,血清DA浓度降低,海马CA1区神经元计数降低,P2Y1R、dysbindin-1表达上调,AMPK表达下调,p-SIRT1/SIRT1比值降低,p-NF-κB p65/NF-κB p65比值升高(P＜0.05);与模型组比较,PNS低剂量组、PNS中剂量组和PNS高剂量组逃避潜伏期缩短,穿越原平台位置次数增多,目标象限停留时间延长,血清IL-1β、IL-6和TNF-α浓度降低,血清DA浓度升高,海马CA1区神经元计数升高,P2Y1R、dysbindin-1表达下调,AMPK表达上调,p-SIRT1/SIRT1 比值升高,p-NF-κB p65/NF-κB p65 比值降低(P＜0.05);与 PNS 高剂量组比较,PNS高剂量+compound C组逃避潜伏期延长,穿越原平台位置次数减少,目标象限停留时间缩短,血清IL-1β、IL-6和TNF-α浓度升高,血清DA浓度降低,海马CA1区神经元计数降低,P2Y1R、dysbindin-1表达上调,AMPK表达下调,p-SIRT1/SIRT1比值降低,p-NF-KB p65/NF-KB p65比值升高(P＜0.05).结论 PNS减轻机械通气大鼠脑损伤的机制可能与激活AMPK-SIRT1-NF-κB信号通路有关.
Abstract
Objective To evaluate the role of the AMP-activated protein kinase(AMPK)-silent information regulator 1(SIRT1)-nuclear factor-κB(NF-κB)signaling pathway in reduction of brain injury by panax notoginseng saponins(PNS)in mechanically ventilated rats.Methods Seventy-two SPF-grade male Sprague-Dawley rats,aged 10 weeks,weighing 357-377 g,were divided into 6 groups(n=12 each)by a random number table method:sham operation group,model group,PNS low dose group,PNS medium dose group,PNS high dose group,and PNS high dose+compound C group.PNS 12.5,25 and 50 mg/kg were intraperitoneally injected in PNS low dose group,PNS medium dose group and PNS high dose group,respectively.In PNS high dose+compound C group,PNS 50 mg/kg was intraperitoneally injected,and 10 min later compound C 0.2 mg/kg was injected via the tail vein.Normal saline 10 ml/kg was intraperitone-ally injected in sham operation group and model group.Drugs or normal saline was injected at 30 min before mechanical ventilation in each group.Mechanical ventilation model:The animals were mechanically ventila-ted for 6 h,with ventilation frequency 40 times/min,tidal volume 40 ml/kg in model group;The animals were mechanically ventilated for 6 h,with tidal volume 10 ml/kg in sham operation group.Morris water maze test was used to detect the learning and memory function of rats,the concentrations of serum interleukin-1beta(IL-1β),IL-6,tumor necrosis factor-alpha(TNF-α)and dopamine(DA)were detected by enzyme-linked immunosorbent assay,the neuronal counts in the hippocampal CA1 region were determined by Nissl staining,and the expression of P2Y1 purine receptor(P2Y1R),dysbindin-1 and AMPK in hippocampal CA1 region was detected by Western blot.The phosphorylated SIRT1(p-SIRT1)to SIRT1 ratio and phos-phorylated NF-κB p65(p-NF-κB p65)to NF-κB ratio in hippocampal CA1 region was calculated.Results Compared with sham operation group,the escape latency was significantly prolonged,the number of crossing the original platform quadrant was reduced,the time of staying at the target platform quadrant was shortened,the serum concentrations of IL-1β,IL-6 and TNF-α were increased,the serum DA concentration was decreased,the nerve count in hippocampal CA1 region was decreased,the expression of P2Y1R and dysbindin-1 was up-regulated,the expression of AMPK was down-regulated,the p-SIRT1/SIRT1 ratio was decreased,and the p-NF-KB p65/NF-KB p65 ratio was increased in model group(P＜0.05).Compared with model group,the escape latency was significantly shortened,the number of crossing the original platform quadrant was increased,the time of staying at the target platform quadrant was prolonged,the serum con-centrations of IL-1β,IL-6 and TNF-α were decreased,the serum DA concentration was increased,the nerve count in hippocampal CA1 region was increased,the expression of P2Y1R and dysbindin-1 was down-regulated,the expression of AMPK was up-regulated,the p-SIRT1/SIRT1 ratio was increased,and the p-NF-κB p65/NF-κB p65 ratio was decreased in PNS low dose group,PNS medium dose group and PNS high dose group(P＜0.05).Compared with PNS high dose group,the escape latency was significantly pro-longed,the number of crossing the original platform quadrant was reduced,the time of staying at the target platform quadrant was shortened,the serum concentrations of IL-1β,IL-6 and TNF-α were increased,the serum DA concentration was decreased,the nerve count in hippocampal CA1 region was decreased,the ex-pression of P2Y1R and dysbindin-1 was up-regulated,the expression of AMPK was down-regulated,the p-SIRT1/SIRT1 ratio was decreased,and the p-NF-κB p65/NF-KB p65 ratio was increased in PNS high dose+compound C group(P＜0.05).Conclusions The mechanism by which PNS reduces brain injury may be related to activation of the AMPK-SIRT1-NF-κB signaling pathway in mechanically ventilated rats.
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