甲基-β-环糊精对内皮素-1诱导大鼠颈总动脉血管平滑肌细胞胞内钙浓度变化的影响

Effects of methyl-β-cyclodextrin on endothelin-1-induced intracellular calcium cation concentration in rat internal carotid artery vascular smooth muscle cells

目的研究甲基-β-环糊精(methyl-β-cyclodextrin,MβCD)对内皮素-1(endothelin-1,ET-1)诱导大鼠颈总动脉血管平滑肌细胞(vascular smooth muscle cells,VSMCs)胞内钙离子浓度([Ca2+]i)变化的影响。方法原代培养大鼠颈总动脉平滑肌细胞,通过形态学和免疫荧光染色鉴定。再通过炎症因子TNF-α(100μg/L)和MβCD(5 mmol/L)的不同处理将细胞分为4组:PSS液+ET-1组(A组)、PSS液+MβCD+ET-1组(B组)、PSS液+TNF-α+ET-1组(C组)、PSS液+TNF-α+MβCD+ET-1组(D组)。最后各组细胞经Furo-2/AM染色后,采用TILL荧光钙成像系统检测活细胞在ET-1(100 nmol/L)刺激下胞内钙荧光强度的变化情况,R(F 340 nm/F 380 nm)值代表单细胞胞内钙荧光强度比值。结果TILL荧光钙成像系统检测结果显示A^D组细胞静息状态荧光强度R0值分别为(0.591 6±0.082 0)、(0.575 3±0.079 0)、(0.655 1±0.083 7)、(0.610 5±0.080 4),组间比较无统计学差异(P>0.05)。各组细胞经ET-1(100 nmol/L)刺激后胞内钙荧光强度分别较静息状态上升:A组上升(115±17)%,B组上升(67±15)%,与A组比较差异有统计学意义(P<0.05);C组上升(279±21)%,与A组比较差异有统计学意义(P<0.01);D组上升(88±16)%,与C组比较差异有统计学意义(P<0.01)。结论 MβCD破坏caveolae结构后明显降低了VSMCs胞内钙离子浓度升高程度,caveolae可能是抑制脑血管痉挛发生的重要靶点。

Objective To determine the effect of methyl-β-cyclodextrin （MβCD） on the change of intracellular calcium cation concentrations induced by endothelin-1 （ET-1） in rat internal carotid artery vascular smooth muscle cells （VSMCs）. Methods The primary cultured rat internal carotid artery VSMCs were identified by morphological observation and immunofluorescence staining. The obtained VSMCs were treated by MβCD （5 mmol/L）, TNF-α（100 μg/L）, and combined treatment, and the cells without treatment served as control. The 4 groups of cells were then stimulated by ET-1 （100 nmol/L） in physiological saline solution （PSS）. After the staining of Ca^2＋-sensitive dye Fluo-2/AM, TILL photonics spectrofluorometry was used to measure the fluorescence ratio （R, F340/F380） for the intracellular calcium changes. Results Without ET-1 stimulation, there was no difference in the fluorescence ratio （R0） of VSMCs in the control, and MβCD, TNF-α and combined treatment cells （0.591 6±0.082 0, 0.575 3±0.079 0, 0.655 1±0.083 7, 0.610 5±0.080 4, P〉0.05）.After 100 nmol /L ET-1 stimulation, the ratio （R） was increased by （115±17）% in the control, and by （67±15）% in the cells treated by MβCD, with significant between the 2 groups （P〈0.05）. The ratio was elevated by （279±21）% in the cells treated by TNF-α and by （88±16）% in those with combined treatment after the same stimulation. Significant differences were seen between the cells treated by TNF-α and control P〈0.01）, and between the combined treatment cells and the cells treated by TNF-α （P〈0.01）. Conclusion MβCD destroys caveolae structure and then obviously reduces the ET-1-increased intracellular calcium cation concentration in rat internal carotid artery VSMCs, and the caveolae may be a potential target for preventing cerebral vasospasm.