异丙酚对新生大鼠海马生存素和半胱氨酸天冬氨酸蛋白酶3表达的影响

Effects of propofol on expression of hippocampal survivin and Caspase-3 in newborn rats

目的观察异丙酚麻醉对新生大鼠海马神经元形态结构、生存素（Survivin）和半胱氨酸天冬氨酸蛋白酶3（Caspase．3）基因表达的影响。方法将体重10-15g的7日龄雄性sD大鼠100只，以数字随机法随机分为4组，每组25只。对照组（A组）不注射任何药物，其他3组分别腹腔注射异丙酚50mg／kg（B组）、异丙酚100mgJkg（C组）或异丙酚200mg／kg（D组）。血气分析仪检测大鼠动脉血呼吸和代谢指标的变化，透射电镜观察海马CAl区神经元细胞形态学变化，Fluoro-Jade8（FJB）荧光染色法检测海马CAl区变性、坏死、凋亡的神经元，半定量逆转录一聚合酶链反应（RT-PCR）和Westernblot技术检测海马组织中Survivin和Caspase-3基因mRNA和蛋白的表达。结果麻醉维持时间：D组〉C组〉B组；各组大鼠动脉血pH、氧分压（PaO2）、二氧化碳分压（PaCO，）、HCO；、碱剩余（BE）、氧饱和度（SaO2）之间比较差异无统计学意义（P〉0．05）。A组、B组海马神经元结构基本正常，C组神经元细胞核肿胀，D组核碎裂、染色质边集甚至出现凋亡小体；A组、B组、c组、D组大鼠海马CAl区FJB阳性细胞数分别为（0．6±0．3）、（2．5±1．3）、（7．1±2．3）、（9．4±2．6）．B组、C组、D组与A组比较差异有统计学意义（P〈0．05），而且FJB阳性细胞数：D组〉C组〉B组。A组、B组、C组海马组织Caspase-3mRNA的表达量分别为（0．78±0．12）、（0．84±0．17）、（0．89±0．19），Caspase-3蛋白的表达量分别为（0．22±0．05）、（0．264±0．07）、（0．214±0．06）；A组、B组、C组SurvivinmRNA的表达量分别为（0．56±0．12）、（0．584±0．15）、（0．534±0．16），Survivin蛋白的表达量分别为（0．24±0．07）、（0．214±0．05）、（0．234±0．06）；A组、B组、C组Caspase-3和SurvivinmRNA和蛋白的表达差异无统计学意义（P〉0．05）；然而，D组海马组织Caspase-3mRNA和蛋白的表达量分别为（1．21±0．14）、（0．42±0．12），高于A组、B组、c组（P〈0．05）；D组海马组织SurvivinmRNA和蛋白的表达量分别为（0．41±0．11）、（0．12±0．03），低于A组、B组、C组（P〈0．05）。结论较大剂量异丙酚可以通过非麻醉缺氧作用促进海马神经元细胞的变性、坏死和凋亡，使海马组织Caspase-3的活性增强而Survivin的表达减少。

Objective Intravenous anesthetics, such as propofol, are widely used in general anesthesia. Neurodegeneration and neurocognitive impairment after exposure to propofol in neonatal rats have raised concerns regarding the safety of pediatric anesthesia. We examined the effects of neonatal propofol exposure on brain cell viability, as well as expression of hippocampal survivin and Caspase-3 mRNA amt protein. Methods One hundred male Sprague-Dawley rats aged 7 d that were weighed 10-15 g were randomly divided into 4 groups （n =25 each group）. Group A： the rats were injected with no drugs. Group B： the rats were intraperitoneally injected with 50 mg/kg propofol. Group C： the rats were first intraperitoneally injected with 50 mg/kg propofol and another 50 mg/kg propofol was used when the dynamic response of rats appeared again. Group D： the rats were first intraperitoneally injected with 50 mg/kg propofol and another 50 mg/kg propofol was used three times once the dynamic response of rats appeared. Tostudy the effects of propofol exposure on respiratmy and metabolic function, arterial blood was aspirated from the left ventricle of neonatal rats 2 h after discontinuation of propofol, pH, PaO2, PaCO2, HCO3- , BE and SaO2 were detected by blood gas analyzer. Moreover, to examine the effects of propofol exposure on short- term cellular viability, the uhrastructure of neurons was observed by transmission electron microscope and Fluoro-Jade B （FJB） staining was performed to examine neuronal degeneration in hippocampal CA1 region of neonatal rats. Survivin and Caspase-3 mRNA and protein expression in hippocampus were detected by semi- quantitative reverse transcription polymerase chain reaction （RT-PCR） and Western blotting 2 h after discontinuation of propofol. Results The time of anesthesia maintaince in newborn rats was the longest in Group D and the time of anesthesia maintaince in Group C was longer than that in Group B. Two hours after discontinuation of propofol, pH, PaO2 , PaCO2, HCO[ , BE and SaO2 of arterial blood in rats were not significantly different among groups A, B, C and D （P 〉 0. 05 ）. The structure of hippocampal neurons was normal in Group A and Group B while 100 mg/kg propofol resulted in nuclear blebbing and 200 mg/kg propofol led to nuclear fragmentation, chromatin condensation and apoptotic bodies. Cellular degeneration, as measured by Fluoro-Jade B staining, significantly increased in hippocampal CA1 region in the anesthesia groups compared with littermates in the no anesthesia group. FJB-positive stained degenerative neurons in groups B, C and D were （2. 5± 1.3 ）, （7.1 ± 2. 3 ） and （ 9.4 ± 2.6）, which were different from that in Group A （0. 6 ± 0. 3 ） （ P 〈 0. 05 ）. Moreover, the number of FJB-positive neurons was the highest in GroupD, that in Group C was more than that in Group B. At the same time point, apoptosis was measured by expression of Caspase-3 and Survivin mRNA and protein in hippocampus of rats. Caspase-3 mRNA in groups A, B and C was （0. 78 ±0. 12）, （0. 84 ±0. 17） and （0. 89 ±0. 19）, while Caspase-3 protein in groups A, B and C was （0. 22 ±0. 05）, （0. 26 ±0.07） and （0. 21 ±0. 06）. Survivin mRNA in groups A, B and C was （0.56±0. 12）, （0.58 ±0. 15） and （0.53±0. 16）, while Survivin protein in these 3 groups was （0.24 ±0.07）, （0.21± 0.05 ） and （0. 23 ±0.06 ）. Compared with that in Group A, Caspase-3 and Survivin mRNA and protein were not significantly different among Group B and Group C （ P 〉 0. 05 ）. However, Caspase-3 mRNA and protein in Group D were （1.21 ±0. 14） and （0. 42±0. 12）, which were higher than that in the other 3 groups（P 〈0. 05）. Survivin mRNA and protein in Group D were lower than that in the other 3 groups （ P 〈 0. 05 ）. Conclusions A high dose of propofol exposure may destroy the structure of heroins, induce neurodegeneration, increase Caspase-3 activity and inhibit survivin expression in hippocampus of newborn rats in vivo.