右美托咪定对坏死性小肠结肠炎新生大鼠丙泊酚麻醉后海马神经元的影响

Effect of dexmedetomidine on hippocampal neurons in neonatal rats with necrotizing enterocolitis after propofol anesthesia

目的探讨右美托咪定对坏死性小肠结肠炎新生大鼠丙泊酚麻醉后海马神经元的影响。方法取30只出生1日的新生SD大鼠,制作成坏死性小肠结肠炎模型。断头取脑,分离两侧海马组织,将原代培养6 d的海马神经元随机分为空白对照组(C组)、丙泊酚组(P组)、丙泊酚加右美托咪定组(PD组),每组10只。P组加入丙泊酚,使终浓度为100μmol/L; PD组加入右美托咪定和丙泊酚,使终浓度分别为100 nmol/L和100μrnol/L; C组加入等体积生理盐水。培养6 d,显微镜下观察神经元生长情况,比较3组神经元平均突起总长度。用免疫荧光化学技术标记神经元和星形胶质细胞,比较3组平均荧光密度。细胞经各种处理后,收集并裂解,提取细胞总蛋白,检测蛋白含量(BCA法)。取50μg待测蛋白质,Western blot法测定神经元pAkt和Bcl-2蛋白水平。结果 C组的神经元平均突起总长度与突触素平均荧光密度分别为(133. 20±34. 01)μm与(30. 57±7.85),为3组中最高,显著高于P组的(83.21±18.96)μm与(14.67±4.25)(P<0.05); PD组的神经元平均突起总长度与突触素平均荧光密度分别为(107.11±23.02)μm与(24.42±6.94),与C组比较无统计学意义(P>0.05),但显著高于P组(P<0.05)。C组的Bal-2蛋白与p Akt蛋白水平分别为(0.72±0.21)与(0.85±0.22),均为3组中最高,显著高于P组的(0.19±0.05)与(0.41±0.11)(P<0.05); PD组Bal-2蛋白、pAkt蛋白水平分别为(0.56±0.18)与(0.78±0.19),与C组比较无统计学意义(P> 0.05),但显著高于P组(P<0.05)。结论丙泊酚会影响坏死性小肠结肠炎新生大鼠的海马神经元突起及突触发育,右美托咪定具有一定的神经元保护作用,可抑制丙泊酚对海马神经元突起及突触发育的影响,并且右美托咪定对神经元的保护作用可能与激活P13K-Akt-Bcl-2信号通路从而上调pAkt和Bcl-2蛋白有关。

Objective To investigate the effect of dexmedetomidine on hippocampal neurons in neonatal rats with necrotizing enterocolitis after propofol anesthesia.Methods Total of 30 newborn SD rats born one day were used to make a model of necrotizing enterocolitis.The brain was decapitated and the hippocampus tissues were separated.The hippocampal neurons cultured for 6 days were randomly divided into blank control group group C propofol group group P propofol plus dexmedetomidine group group PD 10 in each group.Group P was added with propofol to a final concentration of 100μmol/L PD group was added with dexmedetomidine and propofol to a final concentration of 100 nmol/L and 100μrnol/L group C was added with an equal volume of normal saline.After 6 days of culture the growth of neurons was observed under a microscope and the average length of the average protrusion of the three groups was compared.Neurons and astrocytes were labeled by immunofluorescence chemistry and the mean optical density of the three groups was compared.After various treatments the cells were collected and lysed to extract total cellular protein and detect protein content BCA method 50μg of the protein to be tested was taken and the levels of pAkt and Bcl-2 protein in neurons were determined by Western blot.Result The average length of the average protrusion of the neurons and the average fluorescence density of synaptophysin in group C were 133.20±34.01μm and 30.57±7.85 which were the highest in the three groups which were significantly higher than 83.21±18.96μm and 14.67±4.25 in the group P P<0.05 the mean total length of neurons and the average fluorescence density of synaptophysin in the PD group were 107.11±23.02μm and 24.42±6.94 respectively there were no statistical significance compared with the C group P >0.05 but significantly higher than those in group P P<0.05.The levels of Bal-2 protein and pAkt protein in group C were 0.72±0.21 and 0.85±0.22 which were the highest in the three groups and significantly higher than 0.19±0.05 and 0.41±0.11 in the group P P<0.05 the levels of Bal-2 protein and pAkt protein in group PD were 0.56±0.18 and 0.78±0.19 there were no significant difference compared with group C P >0.05 but significantly higher than those in group P P<0.05.Conclusion The propofol affects hippocampal neuronal processes and triggering in neonatal rats with necrotizing enterocolitis.Dexmedetomidine has a certain neuroprotective effect which can inhibit the effect of propofol on hippocampal neuron processes and triggering.And the protective effect of dexmedetomidine on neurons may be related to the activation of P13K-Akt-Bcl-2 signaling pathway and up-regulation of pAkt and Bcl-2 proteins.