Background

: Vaccinium bracteatum Thunb. leaves (VBL) are used in traditional herbal medicines to treat various biological diseases. p-coumaric acid (CA), the main active component of VBL, has neuroprotective effects against corticosterone-induced damage in vitro. However, the effects of CA on immobility induced by chronic restraint stress (CRS) in a mouse model and 5-HT receptor activity have not been investigated.

Hypothesis/Purpose

: We investigated the antagonistic effects of VBL, NET-D1602, and the three components of Gαs protein-coupled 5-HT receptors. Additionally, we identified the effects and mechanism of action of CA, the active component of NET-D1602, in the CRS-exposed model.

Methods

: For in vitro analyses, we used 1321N1 cells stably expressing human 5-HT₆ receptors and CHO-K1 expressing human 5-HT₄ or 5-HT₇ receptors cell lines to study the mechanism of action. For in vivo analyses, CRS-exposed mice were orally administered CA (10, 50, or 100 mg/kg) daily for 21 consecutive days. The effects of CA were analyzed by assessing behavioral changes using a forced swim test (FST), measuring levels of hypothalamic-pituitary-adrenal (HPA) axis-related hormones in serum, and acetylcholinesterase (AChE), monoamines, including 5-HT, dopamine, and norepinephrine, using enzyme-linked immunosorbent assay kits. The underlying molecular mechanisms of the serotonin transporter (SERT), monoamine oxidase A (MAO-A), and extracellular signal-regulated kinase (ERK)/protein kinase B (Akt)/mTORC1 signaling were detected using western blotting.

Results

: CA was confirmed to be an active component in the antagonistic effects of NET-D1602 on 5-HT₆ receptor activity through decreases in cAMP and ERK1/2 phosphorylation. Moreover, CRS-exposed mice treated with CA showed a significantly reduced immobility time in the FST. CA also significantly decreased corticosterone, corticotropin-releasing hormone (CRH), and adrenocorticotropic hormone (ACTH) levels. CA enhanced 5-HT, dopamine, and norepinephrine levels in the hippocampus (HC) and prefrontal cortex (PFC) but decreased MAO-A and SERT protein levels. Similarly, CA significantly upregulated the ERK, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), Akt/mTOR/p70S6K/S6 signaling pathways in both HC and the PFC.

Conclusion

: CA contained in NET-D1602 may play the antidepressant effects against CRS-induced depression-like mechanism and the selective antagonist effect of 5-HT₆ receptor.

中文翻译：

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越橘叶提取物对香豆酸对慢性束缚应激小鼠模型的抗抑郁作用及体外血清素6受体拮抗作用

背景

:越橘 bracteatum Thunb。叶子 (VBL) 在传统草药中用于治疗各种生物疾病。对香豆酸 (CA) 是 VBL 的主要活性成分，在体外对皮质酮诱导的损伤具有神经保护作用。然而，尚未研究 CA 对小鼠模型中由慢性束缚应激 (CRS) 诱导的不动和 5-HT 受体活性的影响。

假设/目的

：我们研究了 VBL、NET-D1602 和 Gαs 蛋白偶联 5-HT 受体的三种成分的拮抗作用。此外，我们还确定了 CA（NET-D1602 的活性成分）在 CRS 暴露模型中的作用和机制。

方法

：对于体外分析，我们使用稳定表达人 5-HT ₆受体的 1321N1 细胞和表达人 5-HT ₄或 5-HT ₇受体的 CHO-K1 细胞系来研究作用机制。对于体内分析中，暴露于 CRS 的小鼠连续 21 天每天口服 CA（10、50 或 100 mg/kg）。通过使用强迫游泳试验 (FST) 评估行为变化来分析 CA 的影响，测量血清中下丘脑-垂体-肾上腺 (HPA) 轴相关激素的水平，以及乙酰胆碱酯酶 (AChE)、单胺类，包括 5-HT、多巴胺和去甲肾上腺素，使用酶联免疫吸附测定试剂盒。使用蛋白质印迹法检测了血清素转运蛋白 (SERT)、单胺氧化酶 A (MAO-A) 和细胞外信号调节激酶 (ERK)/蛋白激酶 B (Akt)/mTORC1 信号的潜在分子机制。

结果

:通过降低 cAMP 和 ERK1/2 磷酸化，CA 被证实是 NET-D1602 对 5-HT _{6受体活性拮抗作用的活性成分。}此外，用 CA 处理的 CRS 暴露小鼠在 FST 中的不动时间显着减少。CA 还显着降低皮质酮、促肾上腺皮质激素释放激素 (CRH) 和促肾上腺皮质激素 (ACTH) 水平。CA 增强了海马体 (HC) 和前额皮质 (PFC) 中的 5-HT、多巴胺和去甲肾上腺素水平，但降低了 MAO-A 和 SERT 蛋白水平。同样，CA 显着上调HC 和 PFC 中的ERK、Ca ^{2+ /钙调蛋白依赖性蛋白激酶 II (CaMKII)、Akt/mTOR/p70S6K/S6 信号通路。}

结论

: NET-D1602中含有的CA可能对CRS诱导的抑郁样机制和5-HT ₆受体的选择性拮抗作用发挥抗抑郁作用。