可乐定预防七氟烷对新生小鼠脑神经毒性的机制:与Tau蛋白磷酸化的关系

Mechanism of clonidine preventing sevoflurane-induced neurotoxicity in neonatal mice: relationship with Tau phosphorylation

目的探讨可乐定预防七氟烷对新生小鼠脑神经毒性的机制与Tau蛋白磷酸化的关系。方法SPF级健康C57BL/6野生型新生小鼠72只,6日龄,按随机数字表法分为4组(n=18):正常对照组(C组)、可乐定对照组(CC组)、七氟烷脑神经毒性组(S组)和可乐定预防组(SC组)。在出生后第6、9、12天,S组、SC组吸入3%七氟烷麻醉2 h,在麻醉前分别腹腔注射生理盐水或可乐定1 mg/kg。在出生后第12天麻醉结束后,每组随机取6只小鼠海马组织,采用Western blot法测定Tau-PS202和Tau-PT205位点磷酸化Tau蛋白(AT8)和总Tau蛋白(Tau46)表达,并计算其比值(AT8/Tau46比值)。每组另外12只在出生后第29、30天行新物体识别实验(观察新物体辨别比率);在第31~37天行Morris水迷宫实验(观察逃逸时间和穿越平台次数),行为学实验结束后麻醉下取海马组织,采用Western blot法测定突触后致密物-95(PSD-95)表达。结果与C组相比,S组AT8表达上调,AT8/Tau46比值升高,PSD-95表达下调,穿越平台次数和新物体辨别比率下降(P<0.05);与S组相比,SC组AT8表达下调,AT8/Tau46比值降低,PSD-95表达上调,穿越平台次数和新物体辨别比率明显升高(P<0.05);海马Tau46表达和逃逸时间各组比较差异无统计学意义(P>0.05)。结论可乐定预防七氟烷诱发新生小鼠脑神经毒性的机制可能与抑制Tau蛋白磷酸化有关。

Objective To investigate the relationship between the mechanism of clonidine preventing sevoflurane-induced neurotoxicity and Tau phosphorylation in neonatal mice.Methods Seventy-two SPF healthy newborn C57BL/6 wild-type mice,aged 6 days,were divided into 4 groups(n=18 each)using a random number table method:normal control group(C group),clonidine control group(CC group),sevoflurane-induced neurotoxicity group(S group)and prevention with clonidine group(SC group).Mice inhaled 3%sevoflurane for 2 h daily on postnatal days 6,9 and 12,and normal saline or clonidine 1 mg/kg was intraperitoneally injected before anesthesia in S group and SC group.Six mice were randomly selected from each group after the end of anesthesia on postnatal day 12,and the hippocampal tissues were removed for determination of the expression of the phosphorylated Tau protein(AT8)and total Tau protein(Tau46)at Tau-PS202 and Tau-PT205 sites by Western blot.The ratio of AT8 expression to Tau 46 expression(AT8/Tau46 ratio)was calculated.Another 12 mice in each group underwent novel object recognition test on postnatal days 29-30(the discrimination ratio of novel objects was observed),and the Morris water maze test was performed on postnatal days 31-37(the escape latency and the times of crossing the platform were observed).After the end of the behavioral testing,the hippocampal tissues were harvested under anesthesia to detect the expression of postsynaptic density-95(PSD-95)by Western blot.Results Compared with group C,the expression of AT8 was significantly up-regulated,the ratio of AT8/Tau46 was increased,the expression of PSD-95 was down-regulated,and the number of crossing platforms and novel object discrimination ratio were decreased in group S(P<0.05).Compared with group S,the expression of AT8 was significantly down-regulated,the ratio of AT8/Tau46 was decreased,the expression of PSD-95 was up-regulated,and the number of crossing platforms and novel object discrimination ratio were significantly increased in group SC(P<0.05).There was no significant difference in the expression of Tau46 and escape latency among the four groups(P>0.05).Conclusions The mechanism by which clonidine prevents sevoflurane-induced neurotoxicity in neonatal mice is related to the inhibition of Tau phosphorylation.