脂多糖对大鼠尾核神经元L型电压门控钙通道电流的影响和外源性2-AG的调制作用

目的:探讨内源性大麻素2-花生四烯酰甘油(2-AG)对脂多糖(LPS)损伤的大鼠尾核神经元L型电压门控钙通道(L-VGCC)电流的调制作用及其分子机制。方法:原代培养新生大鼠尾核神经元,分为对照组、LPS组、2-AG组、2-AG+LPS组、SR141716A(CB1受体反向激动剂)+2-AG+LPS组和AM630(CB2受体反向激动剂)+2-AG+LPS组,应用全细胞膜片钳记录2-AG对LPS损伤的大鼠尾核神经元L-VGCC电流的影响;采用Hoechst染色法观察2-AG对LPS诱导的尾核神经元损伤的影响,并用试剂盒测定尾核神经元caspase-3的活性。结果:(1)LPS能增强L-VGCC电流密度,且未影响L-VGCC激活及失活的电学特征;(2)2-AG能抑制LPS增强L-VGCC电流密度的作用;(3)LPS增强L-VGCC电流密度并非是通过CB1和CB2受体起作用的;(4)2-AG本身对尾核神经元L-VGCC电流密度、激活及失活等电流特性均不产生影响;(5)LPS诱导的尾核神经元caspase-3活性增强可被2-AG抑制,CB1受体反向激动剂SR141716A可取消2-AG的这种效应;(6)LPS可诱导尾核神经元表现出典型的凋亡特征,2-AG可使LPS诱导的核固缩细胞数目显著减少。结论:内源性大麻素2-AG可通过调节尾核神经元L-VGCC电流起抗炎作用和保护神经元的效应。

Effect of lipopolysaccharide on L-type voltage-gated calcium channel currents in primarily cultured rat caudate nucleus neurons and its modulation by 2-arachidonoylglycerol

AIM: To explore whether L-type voltage-gated calcium channel (L-VGCC) currents are involved in neurotoxicity of lipopolysaccharide (LPS) and the neuroprotective mechanism of 2-arachidonoylglycerol (2-AG) in caudate nucleus (CN) neurons. METHODS: On the 7th day, the primarily cultured CN neurons were treated with 2-AG, LPS, SR141716A (a CB1 receptor inverse agonist) and AM630 (a CB2 receptor inverse agonist) for 12 h. The L-VGCC currents in the CN neurons were recorded using the whole-cell patch-clamp technique. Hoechst staining was used to detect the effect of 2-AG on neurotoxicity of LPS. Caspase-3 assay kit was used to determine the activity of caspase-3 induced by LPS alone or with 2-AG. RESULTS: LPS enhanced the current density of L-VGCC, but did not influence the activation and deactivation of L-VGCC. 2-AG inhibited the enhancement of L-VGCC currents induced by LPS. The effect of 2-AG on the suppression of L-VGCC currents induced by LPS was not mediated through CB1 or CB2 receptor. 2-AG alone did not affect the L-VGCC currents, activation and deactivation in the CN neurons. 2-AG inhibited LPS-induced elevated activity of caspase-3 in the CN neurons. This effect was inhibited by SR141716A. 2-AG inhibited the cell apoptosis induced by LPS, and this effect was mediated via CB1 receptor. CONCLUSION: Endocannabinoid 2-AG may exert the anti-inflammatory and neuroprotective effects through the modulation of L-VGCC currents in primarily cultured rat CN neurons.