TLR4-p38MAPK-NF-κB信号通路在七氟醚降低老龄大鼠认知功能中的作用

Role of TLR4-p38MAPK-NF-κB signaling pathway in sevoflurane-induced decrease in cognitive function of aged rats

目的评价TLR4-p38MAPK-NF-κB信号通路在七氟醚降低老龄大鼠认知功能中的作用。方法SPF级健康雄性SD大鼠60只,20月龄,体重550~750 g,采用随机数字表法分为5组(n=12):对照组(C组)、七氟醚组(S组)、TAK242+七氟醚组(TS组)、SB202190+七氟醚组(SS组)和PDTC+七氟醚组(PS组)。大鼠麻醉后行气管插管术,连接小动物呼吸机。TS组、SS组和PS组分别侧脑室注射TAK242、SB202190和PDTC 10μl,C组和S组注射含等量DMSO的生理盐水。侧脑室注射后10 min,通过气管导管吸入4%七氟醚6 h,吸入氧浓度30%,吸入氧流量2 L/min。C组吸入空气-氧气混合气体。七氟醚麻醉结束后7 d行Morris水迷宫实验,记录逃避潜伏期和游泳距离。Morris水迷宫实验结束后,处死大鼠取海马组织,分别采用TUNEL法检测神经元凋亡情况,ELISA法检测TNF-α和IL-1β含量,Western blot法检测caspase-3、磷酸化p38MAPK(p-p38MAPK)、总p38MAPK(t-p38MAPK)和胞核NF-κB表达,计算p-p38MAPK/t-p38MAPK比值。结果与C组比较,余4组各时点逃避潜伏期与游泳距离延长,细胞凋亡率、海马TNF-α和IL-1β含量升高,caspase-3、p-p38MAPK和NF-κB表达上调,p-p38MAPK/t-p38MAPK比值升高(P<0.05);与S组比较,TS组、SS组和PS组各时点逃避潜伏期与游泳距离缩短,细胞凋亡率、海马TNF-α和IL-1β含量降低,caspase-3表达下调,TS组和SS组NF-κB表达下调,TS组p-p38MAPK表达下调,p-p38MAPK/t-p38MAPK比值降低(P<0.05);与TS组比较,SS组和PS组各时点逃避潜伏期和游泳距离延长,细胞凋亡率、海马TNF-α和IL-1β含量升高,caspase-3和p-p38MAPK表达上调,p-p38MAPK/t-p38MAPK比值升高,PS组NF-κB表达上调(P<0.05);与SS组比较,PS组NF-κB表达上调(P<0.05)。结论TLR4-p38MAPK-NF-κB信号通路参与了七氟醚降低老龄大鼠认知功能的过程。

Objective To evaluate the role of Toll-like receptor 4(TLR4)-p38 mitogen-assoliated protein kinase(p38MAPK)-nuclear factor kappa B(NF-κB)signaling pathway in sevoflurane-induced decrease in cognitive function of aged rats.Methods Sixty SPF healthy male Sprague-Dawley rats,aged 20 months,weighing 550-750 g,were divided into 5 groups(n=12 each)using a random number table method:control group(C group),sevoflurane group(S group),TAK242 plus sevoflurane group(TS group),SB202190 plus sevoflurane group(SS group),and PDTC plus sevoflurane group(PS group).All the rats were intubated after anesthesia and connected to an animal ventilator.TAK242,SB202190 and PDTC 10μl were injected into the lateral cerebral ventricle in TS,SS and PS groups,respectively,and normal saline containing the equal volume of DMSO was given in C and S groups.Starting from 10 min after lateral cerebral ventricle injection,4%sevoflurane was inhaled for 6 h via the tracheal tube,with the inhaled oxygen concentration 30%and oxygen flow rate 2 L/min.The mixture of air and oxygen was inhaled in C group.The learning and memory ability was assessed by Morris water maze test at 7 days after the end of sevoflurane anesthesia,and the escape latency and swimming distance were recorded.Animals were sacrificed after the end of Morris water maze test,and brains were removed and hippocampi were isolated for determination of neural apoptosis(by TUNEL),contents of tumor necrosis factor-alpha(TNF-α)and interleukin-1beta(IL-1β)in hippocampal tissues(by enzyme-linked immunosorbent assay),and expression of caspase-3,phosphorylated p38MAPK(p-p38MAPK),total p38MAPK(t-p38MAPK)and NF-κB in nucleus(by Western blot).The apoptosis rate and p-p38MAPK/t-p38MAPK ratio were calculated.Results Compared with C group,the escape latency and swimming distance were significantly prolonged at each time point,the apoptosis rate and contents of TNF-αand IL-1βwere increased,the expression of caspase-3,p-p38MAPK and NF-κB was up-regulated,and p-p38MAPK/t-p38MAPK ratio was increased in the other four groups(P<0.05).Compared with S group,the escape latency and swimming distance were significantly shortened at each time point,the apoptosis rate and contents of TNF-αand IL-1βwere decreased,and the expression of caspase-3 was down-regulated in TS,SS and PS groups,the expression of NF-κB was significantly down-regulated in TS and SS groups,and the expression of p-p38MAPK was significantly down-regulated,and p-p38MAPK/t-p38MAPK ratio was decreased in TS group(P<0.05).Compared with TS group,the escape latency and swimming distance were significantly prolonged at each time point,the apoptosis rate and contents of TNF-αand IL-1βwere increased,the expression of caspase-3 and p-p38MAPK was up-regulated,and p-p38MAPK/t-p38MAPK ratio was increased in SS and PS groups,and the expression of NF-κB was significantly up-regulated in PS group(P<0.05).The expression of NF-κB was significantly up-regulated in PS group when compared with SS group(P<0.05).Conclusion TLR4-p38MAPK-NF-κB signaling pathway is involved in sevoflurane-induced decrease in cognitive function of aged rats.