贯叶金丝桃素和金丝桃苷下调大鼠 C6 神经胶质瘤细胞β1-肾上腺素能受体。
Downregulation of β1 -adrenergic receptors in rat C6 glioblastoma cells by hyperforin and hyperoside from St John's wort.
机构信息
Institute of Biochemistry and Molecular Biology, Rheinische Friedrich-Wilhelm University, Bonn, Germany.
出版信息
J Pharm Pharmacol. 2013 Jun;65(6):907-15. doi: 10.1111/jphp.12050. Epub 2013 Mar 18.
OBJECTIVES
While the use of St John's wort extracts as treatment for mild to moderate depression is well established the mode of action is still under investigation. Individual constituents of St John's wort extract were tested for possible effects on the β1 AR density and a subsequent change in downstream signalling in rat C6 glioblastoma cells.
METHODS
The effect of compounds from St John's wort extract on the downregulation of β1 -adrenergic receptor-GFP fusion proteins (β1 AR-green fluorescent protein (GFP)) of transfected rat C6 gliobastoma cells (C6-β1 AR-GFP) was investigated by means of confocal laser scanning microscopy (LSM). The influence on the lateral mobility of β1 AR-GFP in C6-β1 AR-GFP was investigated by fluorescence correlation spectroscopy. The formation of second messenger was determined by c-AMP-assay.
KEY FINDINGS
Confocal LSM revealed that pretreatment of cells with 1 μm of hyperforin and hyperoside for 6 days, respectively, led to an internalization of β1 AR-GFP under non-stimulating conditions. Observation by fluorescence correlation spectroscopy showed two diffusion time constants for control cells, with τdiff1 = 0.78 ± 0.18 ms and τdiff2 = 122.53 ± 69.41 ms, similarly distributed. Pretreatment with 1 μm hyperforin or 1 μm hyperoside for 3 days did not alter the τdiff values but decreased the fraction of τdiff1 whereas the fraction of τdiff2 increased significantly. An elevated level of β1 AR-GFP with hindered lateral mobility was in line with β1 AR-GFP internalization induced by hyperforin and hyperoside, respectively. A reduced β1 -adrenergic responsiveness was assumed for C6 gliobastoma cells after pretreatment for 6 days with 1 μm of both hyperforin and hyperoside, which was confirmed by decreased cAMP formation of about 10% and 5% under non-stimulating conditions. Decrease in cAMP formation by 23% for hyperforin and 15% for hyperoside was more pronounced after stimulation with 10 μm dobutamine for 30 min.
CONCLUSIONS
The treatment of C6 gliobastoma cells with hyperforin and hyperoside results in a reduced β1 AR density in the plasma membrane and a subsequent reduced downstream signalling.
目的
尽管圣约翰草提取物被广泛用于治疗轻度至中度抑郁症，但作用机制仍在研究中。本研究检测了圣约翰草提取物中的单个成分对大鼠 C6 神经胶质瘤细胞β1 肾上腺素能受体密度的可能影响及其下游信号转导的后续变化。
方法
通过共聚焦激光扫描显微镜（LSM）研究圣约翰草提取物中的化合物对转染大鼠 C6 神经胶质瘤细胞（C6-β1 AR-GFP）中β1 -肾上腺素能受体-GFP 融合蛋白（β1 AR-绿色荧光蛋白（GFP））下调的影响。通过荧光相关光谱法研究β1 AR-GFP 在 C6-β1 AR-GFP 中的侧向流动性的影响。通过 c-AMP 测定法确定第二信使的形成。
主要发现
共聚焦 LSM 显示，分别用 1μM 金丝桃素和金丝桃苷预处理细胞 6 天，可在非刺激条件下导致β1 AR-GFP 的内化。荧光相关光谱观察显示，对照细胞有两个扩散时间常数，τdiff1 = 0.78±0.18ms，τdiff2 = 122.53±69.41ms，分布相似。用 1μM 金丝桃素或 1μM 金丝桃苷预处理 3 天不会改变τdiff 值，但降低了τdiff1 的分数，而τdiff2 的分数显著增加。β1 AR-GFP 水平升高且侧向流动性受阻，与金丝桃素和金丝桃苷分别诱导的β1 AR-GFP 内化一致。用 1μM 金丝桃素和金丝桃苷预处理 6 天后，C6 神经胶质瘤细胞的β1 肾上腺素能反应性降低，这通过在非刺激条件下 cAMP 形成减少约 10%和 5%得到证实。用 10μM 多巴酚丁胺刺激 30min 后，金丝桃素和金丝桃苷的 cAMP 形成分别减少 23%和 15%更为明显。
结论
用金丝桃素和金丝桃苷处理 C6 神经胶质瘤细胞可导致质膜中β1 肾上腺素能受体密度降低，随后下游信号转导减少。
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