盐酸小檗碱对HaCaT细胞内游离钙离子及线粒体膜电位的影响

Berberine-induced Changes of Mitochondria Membrane Potential and Free Intracellular Calcium of HaCaT Cells

目的探讨盐酸小檗碱对角质形成细胞的作用机制。方法用激光扫描共聚焦显微镜及其技术观察盐酸小檗碱作用前后,单个活细胞内Fluo-3/AM及罗丹明-123(Rhodamine-123)荧光强度的变化,推测盐酸小檗碱对HaCaT细胞内游离钙离子和线粒体膜电位的作用及能量代谢的影响。结果对Fluo-3/AM及罗丹明-123标记的HaCaT细胞观察发现:静息状态下,Fluo-3/AM的荧光强度较弱,细胞内呈局限性分布;罗丹明-123的荧光强度在细胞周边最高,核区的荧光强度最低。PBS加入后,细胞内Fluo-3/AM及罗丹明-123荧光强度没有明显变化。盐酸小檗碱呈浓度依赖性持续增强HaCaT细胞内Fluo-3/AM荧光强度,但迅速降低罗丹明-123荧光强度。结论盐酸小檗碱可能通过增加HaCaT细胞内游离钙离子的浓度,降低HaCaT细胞线粒体膜电位,达到其抑制角质形成细胞增殖的作用。

Objective To study the effects on berberine hydrochloride on the mitochondria membrane potential and free intracellular calcium of HaCaT cells, and elucidate the mechanism of action of berberine on keratinocytes. Methods Rhodamine-123 fluorescence (very sensitive to mitochondria membrane potential) and Fluo-3/AM fluorescence (suitabe to detect free intracellular calcium in single HaCaT cell) were measured by laser scanning confocal technique. Results Fluo-3/AM fluorescence intensity of HaCaT cells was persistently increased after treating with berberine at concentrations of 5 × 10-5M, 2.5 × 10-5M and 1.25 × 10-5M, and significant differences were observed as compared with the PBS control. The intensity of rhodamine-123 fluorescence in HaCaT cells was decreased immediately when exposed to berberine, with significant difference from that of the PBS control. Conclusions It is suggested that berberine could increase free intracellular calcium and decrease mitochondria membrane potential of HaCaT cells, induce overload of intracellular calcium, influence energy metabolism, and then inhibit the proliferation of keratinocytes.