缝隙连接阻断剂18α-甘草次酸对大鼠血管平滑肌细胞增殖的作用

Roles of Gap Junction Blocker 18α-Glycyrrhetinic Acid in Proliferation of Vascular Smooth Muscle Cells from Rats

为探讨缝隙连接是否参与抑制大鼠血管平滑肌细胞的增殖及可能的分子机制,本研究以原代及传代培养大鼠胸主动脉平滑肌细胞为模型,实验分2组:对照组、缝隙连接阻断剂18α-甘草次酸(18α-Glycyrrhetinic acid,18α-GA)组。MTT法及流式细胞术检测细胞的增殖活性,染料示踪分子传递法(划痕标记染料传输法)检测细胞的缝隙连接功能,Western blotting法检测细胞中缝隙连接蛋白40(Cx40)和43(Cx43)表达。结果显示:(1)与对照组相比,18α-GA组MTT法测得的A570值及细胞周期S期比例均降低(P<0.01),细胞增殖活性减弱;(2)与对照组相比,18α-GA组罗氏黄荧光染料传递百分数显著降低,缝隙连接功能明显减弱(P<0.01);(3)与对照组相比,18α-GA组Cx40和总Cx43蛋白表达无显著差异(P>0.05),磷酸化Cx43与非磷酸化Cx43的比值显著降低(P<0.01)。由此可知,18α-GA可能主要通过下调血管平滑肌细胞磷酸化Cx43蛋白表达,引起缝隙连接通讯功能减弱,从而抑制了血管平滑肌细胞的增殖。

To investigate whether gap j unction participates in inhibition of proliferation of vascular smooth muscle cells （VSMC） from rats,we sampled the thoracic aorta from the rats. The primary VSMC were isolated and cultured in vitro. The cells were divided into control group and 18α- GA group. The proliferation of VSMC was observed by the methods of MTT and flow cy tometry. The protein levels of Cx43 and Cx40 in the cells were measured by Western blotting. The method of molecular dye transfer（scrape dye transfer method） was applied to detect the function of gap junction in VSMC. The results were as follows： compared with control group,the A570, value detected by MTT and the percentage of S-phase detected by cell cycle in 18α-GA group obviously decreased （P〈0.01）, indicating that the proliferation of the cells decreased. Compared with control group,the protein expression of Cx40 and total Cx43 in 18α-GA group had no significant difference （P〉0.05）. However, the ratio of phosphorylated- and nonphosphorylated- Cx43 protein in 18α-A group significantly decreased （P〈0.01）. Compared with con- trol group,the percentage of dye transfer decreased in 18α-A group （P〈0.01）,the gap junction function of 18α-GA group significantly weakened. We conclude that 18α-A decreases the function of gap junction to inhibit the proliferation of VSMC through down-regulation of the expression of phosphorylated Cx43 protein.