孕酮减轻七氟醚诱导原代海马神经元损伤

Progesterone attenuates sevoflurane-induced neuronal injury in primary hippocampal neurons

目的探究孕酮是否能够减轻七氟醚诱导的原代海马神经元损伤及其可能机制。方法取新生24 h内的SD乳鼠,提取海马神经元培养至第7天后随机分为对照组(C组)、七氟醚组(S组)、孕酮+七氟醚组(P组)和醋酸乌利司他+孕酮+七氟醚组(U组)。C组神经元加入溶剂二甲基亚砜(DMSO)后正常培养;S组加入DMSO后行七氟醚处理;P组加入孕酮1μmol/L预处理1h后行七氟醚处理;U组加入醋酸乌利司他1μmol/L预处理1 h后,余同P组。采用CCK-8法检测神经元细胞活力,采用TUNEL染色检测细胞凋亡率,采用Western blot法测定蛋白激酶B(Akt)和磷酸化蛋白激酶B(p-Akt)蛋白含量,在倒置相差显微镜下观察各组细胞形态改变。结果与C组比较,S组细胞活力明显减弱、细胞凋亡率明显升高、p-Akt蛋白含量和p-Akt/Akt比值明显下降(P<0.01);与S组比较,P组细胞活力明显增强、细胞凋亡率明显降低、p-Akt蛋白含量和p-Akt/Akt比值明显升高(P<0.01);与P组比较,U组细胞活力明显减弱、细胞凋亡率明显升高、p-Akt蛋白含量和p-Akt/Akt比值明显下降(P<0.01)。形态学上,C组细胞形态正常,S组和U组细胞表现出凋亡形态,而P组细胞形态基本正常。结论孕酮可通过孕酮受体上调Akt蛋白磷酸化水平减轻七氟醚诱导的原代海马神经元损伤。

Objective To explore whether progesterone can attenuate sevoflurane-induced neuronal injury in primary hippocampal neurons and its possible mechanism. Methods Hippocampal neurons were isolated from postnatal day 1 SD rats and were cultured in vitro for 7 d. Cells were randomly divided into four groups: control group(group C),sevoflurane group(group S), progesterone+sevoflurane group(group P)and ulipristal acetate+progesterone+ sevoflurane group(group U). The neurons in group C received no treatment but the addition of solvent dimethyl sulfoxide(DMSO). Group S was treated with sevoflurane after adding DMSO. Group P was pretreated with 1 μmol/L of progesterone for 1 h, followed by exposure of sevoflurane. Group U was pretreated with 1 μmol/L ulipristal acetate for 1 h before adding progesterone and exposure of sevoflurane. Then cell viability and apoptosis were respectively assessed by CCK-8 and TUNEL staining. Western blot analysis was performed to detect the expression of p-Akt and Akt. Morphological changes of cells were observed under inverted phase contrast microscopy. Results Compared with group C, the cell viability of group S was significantly lower, the apoptotic rate was significantly higher, and the expression of p-Akt protein and p-Akt/Akt were significantly decreased(P<0.01). Compared with group S, the cell viability of group P was significantly higher, the apoptotic rate was significantly lower, and the expression of p-Akt protein and p-Akt/Akt were significantly increased(P<0.01). Compared with group P, the cell viability of group U was significantly lower, the apoptotic rate was significantly higher, and the expression of p-Akt protein and p-Akt/Akt were significantly decreased(P<0.01). Cells in group C and group P had normal or basically normal morphology, while cells in group S and U manifested typical apoptosis character. Conclusion Progesterone can attenuate sevoflurane-induced neuronal injury in primary hippocampal neurons by binding to progesterone receptors and up-regulating the expression of p-Akt.