School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China; Neurology Department, Kunshan Hospital Affiliated to Nanjing University of Chinese Medicine, Kunshan, China.
School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Phytomedicine. 2023 Jul 25;116:154888. doi: 10.1016/j.phymed.2023.154888. Epub 2023 May 18.
Zhi-Zi-Chi-Tang (ZZCT) is an effective traditional Chinese medicinal formula. ZZCT has been used for the treatment of depression for centuries. Its clinical efficacy in relieving depression has been confirmed. However, the molecular mechanisms of ZZCT regarding neuroplasticity in the pathogenesis of depression have not yet been elucidated.
The present study aimed to examine the effects of ZZCT on neuroplasticity in mice exposed to chronic unpredictable mild stress (CUMS), and to explore the underlying molecular mechanisms.
For this purpose, a murine model of depression was established using the CUMS procedure. Following the intragastric administration of ZZCT or fluoxetine, classic behavioral experiments were performed to observe the efficacy of ZZCT as an antidepressant. Immunofluorescence was used to label and quantify microtubule-associated protein (MAP2) and postsynaptic density protein (PSD95) in the hippocampus. Golgi staining was applied to visualize the dendritic spine density of neurons in the hippocampi. Isolated hippocampal slices were prepared to induce long-term potentiation (LTP) in the CA1 area. The hippocampal protein expression levels of glycogen synthase kinase-3β (GSK-3β), p-GSK-3β (Ser9), cAMP response element binding protein (CREB), p-CREB (Ser133), brain-derived neurotrophic factor (BDNF) and 14-3-3ζ were detected using western blot analysis. The interaction of 14-3-3ζ and p-GSK-3β (Ser9) was examined using co-immunoprecipitation. LV-shRNA was used to knockdown 14-3-3ζ by an intracerebroventricular injection.
ZZCT (6 g/kg) and fluoxetine (20 mg/kg) alleviated depressive-like behavior, restored hippocampal MAP2 PSD95 intensity, and reversed the dendritic spine density of hippocampal neurons and LTP in the CA1 region of mice exposed to CUMS. Both low and high doses of ZZCT (3 and 6 g/kg) significantly promoted the binding of 14-3-3ζ to p-GSK-3β (Ser9) in the hippocampus, and ZZCT (6 g/kg) significantly promoted the phosphorylation of GSK-3β Ser9 and CREB Ser133 in the hippocampus. ZZCT (3 and 6 g/kg) upregulated hippocampal BDNF expression in mice exposed to CUMS. LV-sh14-3-3ξ reduced the antidepressant effects of ZZCT.
ZZCT exerted antidepressant effects against CUMS-stimulated depressive-like behavior mice. The knockdown of 14-3-3ζ using lentivirus confirmed that 14-3-3ζ was involved in the ZZCT-mediated antidepressant effects through GSK-3β/CREB/BDNF signaling. On the whole, these results suggest that the antidepressant effects of ZZCT are attributed to restoring damage by neuroplasticity enhancement via the 14-3-3ζ/GSK-3β/CREB/BDNF signaling pathway.
栀子豉汤（ZZCT）是一种有效的中药方剂。ZZCT 已被用于治疗抑郁症已有几个世纪。其在缓解抑郁症方面的临床疗效已得到证实。然而，ZZCT 对抑郁症发病机制中神经可塑性的分子机制尚未阐明。
本研究旨在探讨 ZZCT 对慢性不可预测轻度应激（CUMS）小鼠神经可塑性的影响，并探讨其潜在的分子机制。
为此，采用 CUMS 程序建立了抑郁小鼠模型。在给予 ZZCT 或氟西汀灌胃后，进行经典行为实验观察 ZZCT 作为抗抑郁药的疗效。免疫荧光法标记和定量海马中的微管相关蛋白（MAP2）和突触后密度蛋白（PSD95）。高尔基染色用于可视化海马神经元树突棘密度。分离海马切片以诱导 CA1 区长时程增强（LTP）。采用 Western blot 分析检测海马糖原合酶激酶-3β（GSK-3β）、p-GSK-3β（Ser9）、cAMP 反应元件结合蛋白（CREB）、p-CREB（Ser133）、脑源性神经营养因子（BDNF）和 14-3-3ζ 的蛋白表达水平。采用免疫共沉淀法检测 14-3-3ζ 和 p-GSK-3β（Ser9）的相互作用。通过脑室内注射 LV-shRNA 敲低 14-3-3ζ。
ZZCT（6g/kg）和氟西汀（20mg/kg）缓解了抑郁样行为，恢复了 CUMS 暴露小鼠海马 MAP2 PSD95 强度，并逆转了海马神经元树突棘密度和 CA1 区 LTP。低剂量和高剂量的 ZZCT（3 和 6g/kg）均显著促进了海马中 14-3-3ζ 与 p-GSK-3β（Ser9）的结合，而 ZZCT（6g/kg）显著促进了海马中 GSK-3β Ser9 和 CREB Ser133 的磷酸化。ZZCT（3 和 6g/kg）上调了 CUMS 暴露小鼠海马中的 BDNF 表达。LV-sh14-3-3ξ 降低了 ZZCT 的抗抑郁作用。
ZZCT 对 CUMS 刺激的抑郁样行为小鼠具有抗抑郁作用。使用慢病毒敲低 14-3-3ζ 证实 14-3-3ζ 通过 GSK-3β/CREB/BDNF 信号通路参与了 ZZCT 介导的抗抑郁作用。总的来说，这些结果表明，ZZCT 通过增强神经可塑性来恢复损伤，从而发挥抗抑郁作用，其机制可能与 14-3-3ζ/GSK-3β/CREB/BDNF 信号通路有关。
