大黄素甲醚对新生大鼠皮质神经元营养作用的靶点研究

Target Research of Neurotrophic Effect of Physcion on Cultured Neurons in Cerebral Cortex of Newborn Rats

目的探讨大黄素甲醚(physcion,Phy)对新生大鼠皮质神经元营养作用的靶点。方法选用与神经营养作用相关的工具药酪氨酸激酶Trk受体抑制剂K252a、腺苷A2a受体抑制剂ZM241385及蛋白激酶C抑制剂G6976,实验设溶媒对照组、抑制剂组、Phy组、抑制剂+Phy组,四甲基偶氮唑蓝法分析各抑制剂对神经元存活的影响,倒置显微镜下观察神经元突起生长情况,Image-pro plus 6.0软件测量突起长度,分析各抑制剂能否拮抗Phy促进突起生长的作用,初步推断Phy对皮质神经元神经营养作用的靶点。结果溶媒(0.1%DMSO)对照组、K252a(100μmol/L)组、ZM241385(20 nmol/L)组和G6976(100μmol/L)组的神经元活性分别与空白对照组比较,差异均无统计学意义(P>0.01)。K252a(100μmol/L)+Phy组神经元平均突起长度与Phy组比较,差异有统计学意义(P<0.01);而ZM241385(20 nmol/L)+Phy组、G6976(100μmol/L)+Phy组与Phy组比较,神经元平均突起长度差异均无统计学意义(P>0.05)。结论 Phy对新生大鼠神经元的神经营养作用可被酪氨酸激酶Trk受体抑制剂K252a阻断,而腺苷A2a受体抑制剂ZM241385和蛋白激酶C抑制剂G6976的阻断作用则不明显,提示Phy神经营养作用的靶点可能是Trk受体,即Phy通过激动Trk受体而发挥神经营养作用的。

Objective To study the target of neurotrophic effect of physcion on cultured neurons in cerebral cortex of newborn rats.Methods K252a（Trk receptor antagonists）,ZM241385（specific adenosine A2a receptor antagonist） and G6976（PKC antagonist） were used to explore the target of neurotrophic effect of physcion on cortical neurons.Vehicle control group,antagonists group,Phy group and antagonists＋Phy group were established in the study.Methyl thiazolyl tetrazolium（MTT） method was used to analyze the effect of each antagonist on the survival of neurons and the growth of neurites was observed under inverted microscope.Image-pro plus 6.0 was used to test the length of neurons and analyze which antagonist could detain the promoting effect of Phy on the growth of neurites so as to deduce the target of neurotrophic effect of Phy on cultured neurons.Results Vehicle（0.1% DMSO） control group,K252a（100 μmol/L） group,ZM241385（20 nmol/L） group and G6976（100 μmol/L） group showed no significant differences in neuronal viability compared with blank control group（P0.05）.Compared with physcion group,K252a（100 μmol/L） ＋physcion group had significantly shorten the average length of neuritis（P0.01）,but ZM241385（20 nmol/L）＋physcion group and G6976（100 μmol/L）＋physcion group showed no statistically significant difference in average length of cortical neuronal neuritis（P0.05）.Conclusion K252a can restrain the neurotrophic activity of physcion on promoting neurite outgrowth.However,G6976 and ZM241385 show limited restraining effect on physcion,indicating that Trk may be the target of neurotrophic effect of physcion,and that physcion may play its neurotrophic role by activating Trk receptor.