不同浓度丙泊酚麻醉孕中期大鼠对雄性子代学习和记忆功能的影响及其机制

Effects of anesthesia with different concentrations of propofol on male offspring′s learning and memory functions in rats in second trimester of pregnancy and its mechanism

目的探讨不同浓度丙泊酚麻醉孕中期大鼠对雄性子代学习和记忆功能的影响,并分析钙离子-钙调素依赖性蛋白激酶Ⅱ(CaM KⅡ)/环磷腺苷效应元件结合蛋白(CREB)信号通路在其中的作用机制。方法将72只怀孕14 d的雌性大鼠随机分为4组:对照组(C组)大鼠经颈内静脉注射生理盐水后不做处理;丙泊酚低剂量组(L组)、丙泊酚中剂量组(M组)、丙泊酚高剂量组(H组)大鼠分别经颈内静脉注射1.0%、1.5%、2.0%的丙泊酚进行麻醉诱导,然后持续静脉泵注各自浓度的丙泊酚进行麻醉维持,行剖腹探查手术,麻醉时间4 h。取4组出生后30 d的雄性子代进行Morris水迷宫实验以评价其学习、记忆功能。取出生后36 d的M组雄性子代,将腹腔注射CaM KⅡ抑制剂KN-93磷酸盐的子代作为M+KN-93组,腹腔注射生理盐水的子代作为M+saline组,并于出生后43 d行Morris水迷宫实验。于出生后48 d处死各组子鼠并取海马组织,采用Western blot检测生长相关蛋白43(GAP43)、突触后密度蛋白95(PSD95)、B细胞淋巴瘤蛋白-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Caspase3、活化型Caspase3、CaM KⅡ、CREB及磷酸化CREB的表达量,采用尼氏染色法评估M+saline组和M+KN-93组海马组织神经元细胞密度。结果与C组相比,L组、M组、H组雄性子代出生后31~34 d的逃避潜伏期均增加,平台穿越次数均减少,第Ⅰ象限的停留时间均延长,第Ⅱ象限的停留时长均缩短,海马区的Bax、活化型Caspase3、CaM KⅡ表达量均上调,Bcl-2、GAP43、PSD95、磷酸化CREB蛋白表达量均下调(均P<0.05);与L组相比,M组和H组大鼠雄性子代上述指标的改变更明显(均P<0.05),但M组和H组上述指标比较差异均无统计学意义(均P>0.05)。与M+saline组相比,M+KN-93组雄性子代的逃避潜伏期变短,平台穿越次数增加,第Ⅱ象限停留时间增加,海马组织中CaM KⅡ蛋白表达量下调,磷酸化CREB、GAP43、PSD95蛋白表达量上调,神经元细胞密度增加(均P<0.05)。结论使用不同浓度的丙泊酚麻醉孕中期大鼠会导致其雄性子代的学习、记忆功能下降,并伴有海马组织CaM KⅡ蛋白表达上调及CREB激活程度降低,以中、高浓度(1.5%、2.0%)的丙泊酚作用更为明显;抑制CaM KⅡ表达可提高大鼠雄性子代的学习、记忆功能,其作用机制可能与CREB激活有关。

Objective To investigate the effect of anesthesia with different concentrations of propofol on male offspring′learning and memory functions in rats in the second trimester of pregnancy,and to analyze the mechanism underlying calcium/calmodulin-dependent protein kinaseⅡ(CaMKⅡ)/cyclic AMP responsive element binding protein(CREB)signaling pathway functioned in the effect.Methods Seventy-two female rats which had been pregnant for 14 days were randomly divided into four groups:control group(group C)in which the rats were injected with normal saline via internal jugular vein and then did not receive any treatment,low-dose propofol group(group L),medium-dose propofol group(group M)and high-dose propofol group(group H).Rats in group L,group M and group H were injected with 1.0%,1.5%and 2.0%propofol,respectively,via internal jugular vein for anesthesia induction and then received continuous intravenous pumping of propofol at corresponding concentrations for anesthesia maintenance,and an exploratory laparotomy was conducted with an anesthesia duration of four hours.The Morris water maze test was performed on male offspring 30 days after birth,which were selected from the four groups,to assess their learning and memory functions.Among male offspring 36 days after birth which were selected from group M,those intraperitoneally injected with CaMKⅡinhibitor KN-93 phosphate served as M+KN-93 group,whereas those intraperitoneally injected with normal saline served as M+saline group;moreover,these offspring underwent the Morris water maze test 43 days after birth.The offspring of each group were killed 48 days after birth to harvest their hippocampal tissues.Western blot assay was employed to detect the expression of growth-associated protein 43(GAP43),postsynaptic density protein 95(PSD95),B-cell lymphoma-2 protein(Bcl-2),Bcl-2 associated X protein(Bax),Caspase3,cleaved Caspase3,CaMKⅡ,CREB and phosphorylated CREB,and Nissl staining was used to evaluate the density of hippocampal neurons in the M+saline and M+KN-93 groups.Results Compared with male offspring of group C,from day 31 to day 34 after birth,male offspring of group L,group M and group H exhibited increases in the escape latency,decreases in the number of platform crossing,increase in the length of quadrantⅠstay,whereas declines in the length of quadrantⅡstay,up-regulated expression of Bax and cleaved Caspase3,CaMKⅡin hippocampus,and down-regulated expression of Bcl-2,GAP43,PSD95,and phosphorylated CREB proteins(all P<0.05).Compared with male offspring of group L,male offspring of groups M and H exhibited more obvious changes in the aforementioned indexes(all P<0.05),but there were no statistically significant differences in the aforementioned indexes between group M and group H(all P>0.05).Compared with the M+saline group,the M+KN-93 group had a shorter escape latency,an increased number of platform crossing,increased length of quadrantⅡstay,down-regulated expression of hippocampal CaMKⅡprotein,up-regulated expression of phosphorylate CREB,GAP43 and PSD95 proteins,and an increased density of neurons(all P<0.05).Conclusion The administration of anesthesia with different concentrations of propofol to rats in the second trimester of pregnancy will lead to declines in learning and memory functions of their male offspring,accompanied by up-regulation of hippocampal CaMKⅡprotein expression and a decrease in the degree of CREB activation,and propofol at medium and high concentrations(1.5%and 2.0%)exert more significant effects;inhibition of CaMKⅡexpression can improve the learning and memory functions of male offspring of rats,and its mechanism of action may be related to CREB activation.