雌激素对去势合并慢性应激雌性大鼠海马神经元中GPR30表达的影响

Effect of Estrogen on the Expression of GPR30 in the Hippocampal Neurons of Female Rats of Castration with Chronic Stress

目的建立SD大鼠去势合并慢性应激模型,观察海马神经元损伤情况及雌激素膜受体GPR30蛋白在海马中的表达,了解雌激素抗抑郁的作用机制。方法选择雌性SD大鼠,随机分为空白对照组、假手术组、去势+应激模型组和去势+应激+17β-雌二醇共价衍生物(E2-BSA)组,每组12只。旷场实验观察SD大鼠行为变化,尼氏染色观察海马神经元形态变化,免疫组织化学及Western blot检测海马GPR30蛋白阳性表达。结果去势+应激模型组与空白对照组和假手术组相比旷场实验得分均减少(P<0.05),说明去势应激造模成功。去势+应激+E2-BSA组旷场实验得分高于去势+应激模型组(P<0.05),与空白对照组和假手术组相比差异无统计学意义(P>0.05);尼氏染色显示,去势+应激模型组海马神经元细胞排列散乱,尼氏体减少;去势+应激+E2-BSA组大鼠海马神经元细胞形态完整,细胞排列规整,尼氏体较多。与空白对照组、假手术组和去势+应激+E2-BSA组大鼠各区相比较,去势+应激模型组大鼠尼氏体减少(P<0.05),其他三组差异无统计学意义(P>0.05);免疫组化结果显示,与空白对照组、假手术组和去势+应激+E2-BSA组大鼠相比,去势+应激模型组大鼠海马CA1、CA3、DG三区GPR30蛋白阳性表达减少(P<0.05),其他三组差异无统计学意义(P>0.05),各组CA2区GPR30蛋白阳性表达相比差异无统计学意义(P>0.05);Western blot结果显示,去势+应激模型组大鼠GPR30蛋白阳性表达少于空白对照组、假手术组和去势+应激+E2-BSA组(P<0.05),其他三组差异无统计学意义(P>0.05)。结论 E2-BSA干预可减轻去势合并慢性应激SD大鼠海马神经元的损伤,增加GPR30蛋白阳性表达,可能是雌激素保护海马神经元功能的机制之一。

Objective To observe the damage of hippocampal neurons and the expression of G protein-coupled receptor 30（GPR30）in hippocampus after establishing the model of chronic unpredictable stress of castrated rats,exploring the antidepressant mechanism of estrogen. Methods The female SD rats were randomly divided into blank control group, sham group, castration＋stress model group and castration＋stress＋17β-estradiol conjugate（E2-BSA）group,12 rats for each group. The behavior of rats was detected by open field test. Nissl Staining was used to observe the morphology of hippocampal neurons. Immunohistochemistry and Western blot were used to test the positive expression of GPR30 protein of hippocampal neurons. Results The open field score of the castration＋stress model group decreased significantly compared with the blank control group and the sham group（P〈0.05）,which proved that the castrated stress model was successful. The scores of open field test in castration＋stress＋E2-BSA group were higher than that in castration＋stress model group（P〈0.05）, but there was no significant difference compared with the blank control group and the sham group （P〉0.05）. The score of the open field test of the castration＋stress＋E2-BSA group was no significant difference compared with the blank control group and the sham group（P〉0.05）. The score of the open field test of the castration＋stress＋E2-BSA group was higher than that of the castration＋stress model group,and the difference was statistically significant（P〈0.05）. Nissl staining results showed that hippocampal neuronal cells were scattered and Nissl bodies decreased in the castration＋stress group. The hippocampal neurons in the castration＋stress＋E2-BSA group had a complete morphology, regular arrangement of cells,and more Nissl bodies. The hippocampal neurons in the castration＋ stress model group were disorganized and the Nissl bodies decreased.The Nissl bodies in castration＋stress model group decreased significantly compared with the blank control group,sham group and castration＋stress＋ E2-BSA group（P〈0.05）,but there was no significant difference among the other three groups（P〉0.05）. The immunohistochemical results showed that the positive expression of GPR30 protein in hippocampal CA1,CA3 and DG regions was decreased in castrated stress model group compared with the control group, sham group and castration＋stress＋E2-BSA group and the difference was statistically significant（P〈0.05）. There was no significant difference among the other three groups（P〉0.05）. There was no significant difference in the positive expression of GPR30 protein in the hippocampal CA2 region among the four groups （P〉0.05）. The results of Western blot showed that the positive expression of GPR30 protein in castration＋stress model group was lower than that in blank control group,sham group and castration＋stress＋E2-BSA and there was significant difference（P〈0.05）. There was no significant difference among the other three groups （P〉0.05）. Conclusion E2-BSA intervention can reduce the damage of hippocampal neurons and increase the positive expression of GPR30 protein in SD rats after castration and chronic stress,which may be one of the mechanisms of estrogen protecting the hippocampal neurons.