芍药苷对大鼠背根神经元细胞内Ca^(2+)的影响

Effect of paeoniflorin on the calcium concentration in rat dorsal root ganglion neurons

目的建立一种评价芍药苷对大鼠背根神经节神经元细胞内游离Ca^(2+)浓度影响的方法。方法显微解剖获取大鼠背根神经节(DRG),通过胰蛋白酶消化,过筛,用DF-12和抗有丝分裂培养液交替培养纯化,获得原代大鼠DRG神经元细胞,并采用细胞免疫荧光技术测定DRG神经元细胞纯度;采用激光共聚焦显微成像技术,观察细胞内Ca^(2+)荧光强度的变化,并对Ca^(2+)荧光强度变化率进行分析,探讨芍药苷对DRG细胞内游离钙离子浓度及辣椒素受体的影响。结果采用上述方法分离得到的DRG细胞纯度可高达95%以上,辣椒平可通过阻断辣椒素激活的瞬时受体电位通道的作用而抑制细胞内Ca^(2+)的增加。芍药苷表现出与辣椒平类似的作用,可以阻断细胞外Ca^(2+)内流。结论芍药苷可能是通过作用于TRPV1通道,而抑制DRG细胞内Ca^(2+)大量增加,本方法可以用于评价药物对大鼠DRG细胞内Ca^(2+)浓度的影响。

Objective To establish a method to evaluate the effect of paeoniflorin（ PF） on free calcium（ Ca^（2＋） ）concentration in dorsal root ganglion（ DRG） neurons in rats. Methods DRGs were obtained by microdissection,then were digested by trypsin and sieved. The dissociated neurons were alternately cultured and purified with DF-12 and anti-mitotic culture medium to obtain primary rat DRG neurons,and the purity of DRG neurons was measured by immunofluorescence technique. The fluorescence intensity of Ca^（2＋） in DRGs was observed by laser confocal microscopy（ LSCM） imaging,and the changes of fluorescence intensity were analyzed to explore the effects of PF on DRG intracellular free calcium concentration and capsaicin receptor. Results The purity of DRG cells isolated by the above mentioned method was up to 95%.Capsazepine（ CAP） inhibited the increase of intracellular Ca^（2＋） by blocking the capsaicin-activated transient receptor potential vanilloid 1（ TRPV1）. PF exhibited the influx of extracellular Ca^（2＋） . Conclusions We would hypothesize that PF probably inhibits the significant increase of Ca^（2＋） in DRG cells by acting on TRPV1 channel,and the method established in this study can be used to evaluate the effects of drugs on Ca2 ＋concentration in rat dorsal root ganflion neurons.