Cheng Song, Rong Yi, Ma Minjie, Lin Xianguang, Liu Xiangming, Li Chenhong, Yang Xiaofei, Chen Su
Key Laboratory of Cognitive Science of State Ethnic Affairs Commission, Wuhan, Hubei, China.
Hubei Key Laboratory of Medical Information Analysis and Tumor Diagnosis & Treatment, Wuhan, Hubei, China.
J Cell Biochem. 2020 Feb;121(2):1790-1800. doi: 10.1002/jcb.29414. Epub 2019 Oct 23.
To search the modulation mechanism of loureirin B, a flavonoid is extracted from Dracaena cochinchinensis, on tetrodotoxin-resistant (TTX-R) sodium channel in dorsal root ganglion (DRG) neurons of rats. Experiments were carried out based on patch-clamp technique and molecular biological methods. We observed the time-dependent inhibition of loureirin B on TTX-R sodium currents in DRG neurons and found that neither occupancy theory nor rate theory could well explain the time-dependent inhibitory effect of loureirin B on TTX-R sodium currents. It suggested that a second messenger-mediated signaling pathway may be involved in the modulation mechanism. So the cyclin AMP (cAMP) level of the DRG neurons before and after incubation with loureirin B was tested by ELISA Kit. Results showed that loureirin B could increase the cAMP level and the increased cAMP was caused by the enhancement of adenylate cyclase (AC) induced by loureirin B. Immunolabelling experiments further confirmed that loureirin B can promote the production of PKA in DRG neurons. In the presence of the PKA inhibitor H-89, the inhibitory effect of loureirin B on TTX-R sodium currents was reversed. Forskolin, a tool in biochemistry to raise the levels of cAMP, also could reduce TTX-R sodium currents similar to that of loureirin B. These studies demonstrated that loureirin B can modulate the TTX-R sodium channel in DRG neurons via an AC/cAMP/PKA pathway involving the activation of AC and PKA, which also can be used to explain the other pharmacological effects of loureirin B.
为探究从龙血树中提取的黄酮类化合物龙血竭素B对大鼠背根神经节（DRG）神经元中河豚毒素抗性（TTX-R）钠通道的调节机制。实验基于膜片钳技术和分子生物学方法进行。我们观察了龙血竭素B对DRG神经元中TTX-R钠电流的时间依赖性抑制作用，发现占据理论和速率理论均不能很好地解释龙血竭素B对TTX-R钠电流的时间依赖性抑制作用。这表明第二信使介导的信号通路可能参与了调节机制。因此，用ELISA试剂盒检测了龙血竭素B孵育前后DRG神经元的环磷酸腺苷（cAMP）水平。结果显示，龙血竭素B可提高cAMP水平，且cAMP的升高是由龙血竭素B诱导的腺苷酸环化酶（AC）增强所致。免疫标记实验进一步证实，龙血竭素B可促进DRG神经元中蛋白激酶A（PKA）的产生。在存在PKA抑制剂H-89的情况下，龙血竭素B对TTX-R钠电流的抑制作用被逆转。福斯可林是一种用于提高cAMP水平的生物化学工具，它也能像龙血竭素B一样降低TTX-R钠电流。这些研究表明，龙血竭素B可通过AC/cAMP/PKA途径调节DRG神经元中的TTX-R钠通道，该途径涉及AC和PKA的激活，这也可用于解释龙血竭素B的其他药理作用。
