舒更解郁方通过调控PI3K/AKT信号通路对围绝经期抑郁症大鼠星形胶质细胞凋亡作用研究

Study on the effect of Shugeng Jieyu Prescription on apoptosis of astrocytes in perimenopausal depression rats by regulating PI3K/AKT signaling pathway

关键词:  围绝经期抑郁症  PI3K/Akt  细胞凋亡  星形胶质细胞

Key words:  perimenopausal depression  PI3K/Akt  cell apoptosis  astrocytes

摘要:

目的 探讨舒更解郁方通过磷脂酰肌醇3激酶/蛋白激酶B（PI3K/Akt）信号通路调控围绝经期抑郁症（PMD）大鼠星形胶质细胞（AST）凋亡的作用机制。方法 采用大鼠双侧卵巢摘除术结合慢性不可预知温和应激（CUMS）制备PMD模型。将造模成功大鼠随机分为模型组、舒更解郁方低剂量组[2.9 g/（kg·d）]、中剂量组[5.8 g/（kg·d）]、高剂量组[11.6 g/（kg·d）]及逍遥散组，每组10只，并设假手术组作为对照。连续28 d给予应激刺激，并对治疗组大鼠灌胃给药。通过糖水偏好、强迫游泳及新奇摄食实验评估抑郁程度，酶联免疫吸附实验（ELISA）检测海马促肾上腺皮质激素释放激素（CRH）、促肾上腺皮质激素（ACTH）、皮质酮（CORT）浓度，免疫组化检测海马胶质纤维酸性蛋白（GFAP）表达，蛋白免疫印迹法（Western blot）检测海马半胱氨酸蛋白酶-3（Caspase-3）、B淋巴细胞瘤-2基因（Bcl-2）、Bcl-2相关X蛋白（Bax）、磷酸化PI3K（P-PI3K）、磷酸化Akt（P-Akt）蛋白表达，免疫荧光共定位法检测GFAP与Bax、Bcl-2、Caspase-3蛋白的共定位。结果 模型组大鼠表现出抑郁样行为，海马CRH、ACTH、CORT浓度升高，GFAP、Bcl-2、P-PI3K、P-Akt表达降低，Bax、Caspase-3表达增多，Bax阳性细胞和荧光融合增多，Bcl-2阳性细胞和荧光融合减少。舒更解郁方高、中剂量组能显著改善抑郁样行为，降低海马CRH、ACTH、CORT浓度，上调GFAP、Bcl-2、P-PI3K、P-Akt表达，下调Bax、Caspase-3表达，减少Bax阳性细胞及荧光融合，增加Bcl-2阳性细胞及荧光融合（P＜0.01或P＜0.05）。结论 舒更解郁方可激活PI3K/Akt信号通路，抑制海马AST凋亡，从而治疗PMD。

Abstract:

Objective To investigate the mechanism of Shugeng Jieyu Prescription in regulating the apoptosis of astrocytes(AST) in perimenopausal depression(PMD) rats through phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) signaling pathway.Methods PMD model was established by bilateral ovariectomy combined with chronic unpredictable mild stress(CUMS) in rats. The successful model rats were randomly divided into model group, Shugeng Jieyu Prescription low[2.9 g/(kg·d)], medium[5.8 g/(kg·d)], high dose group[11.6 g/(kg·d)] and Xiaoyao Powder group, 10 rats in each group, and sham operation group was set as control. Stress stimulation was given for 28 consecutive days, and the rats in the treatment group were given intragastric administration. The degree of depression was evaluated by sucrose preference test, forced swimming test and novelty feeding test. The concentration of corticotropin-releasing hormone(CRH), adrenocorticotropic hormone(ACTH) and corticosterone(CORT) in hippocampus was detected by ELISA. The expression of glial fibrillary acidic protein(GFAP) protein in hippocampus was detected by immunohistochemistry. The expression of Caspase-3, B-cell lymphoma-2(Bcl-2), BCL-2-associated X protein(Bax), phosphorylated PI3K and phosphorylated Akt protein in hippocampus was detected by Western blot. The co-localization of GFAP with Bax, Bcl-2 and Caspase-3 protein was detected by immunofluorescence co-localization.Results The rats in the model group showed depression-like behavior, the concentration of CRH, ACTH and CORT in hippocampus increased, the expression of GFAP, Bcl-2, P-PI3K and P-Akt decreased, the expression of Bax and Caspase-3 increased, the number of Bax positive cells and fluorescence fusion increased, and the number of Bcl-2 positive cells and fluorescence fusion decreased. The high and middle dose groups of Shugeng Jieyu Prescription could significantly improve depression-like behavior, reduce the concentration of CRH, ACTH and CORT in hippocampus, up-regulate the expression of GFAP, Bcl-2, P-PI3K and P-Akt, down-regulate the expression of Bax and Caspase-3, reduce Bax positive cells and fluorescence fusion, and increase Bcl-2 positive cells and fluorescence fusion(P < 0.01 or P < 0.05).Conclusion Shugeng Jieyu Prescription can activate PI3K/Akt signaling pathway, inhibit the apoptosis of hippocampal AST, and treat PMD.