丹参酮ⅡA对HUVEC细胞间缝隙连接的作用

Effect of Tanshinone ⅡA on Gap Junction Function of Human Umbilical Vein Epithelial Cells

【目的】探讨丹参酮ⅡA(TanshinoneⅡA)对人胎脐静脉上皮细胞(HUVEC)缝隙连接细胞通讯(GJIC)的影响及其机制。【方法】采用四甲基偶氮唑盐(MTT)法检测丹参酮ⅡA对HUVEC细胞生长的影响,荧光显微镜观察及流式细胞仪荧光示踪法分析GJIC功能变化,并与阳性对照药全反式维甲酸(ATRA)比较,采用荧光实时定量PCR(q RT-PCR)法分析Cx37、Cx40 m RNA的表达。【结果】丹参酮ⅡA作用HUVEC 24、48 h的半数致死量(IC50)分别为15、8.5μmol/L,32、64μmol/L丹参酮ⅡA作用HUVEC 48 h有明显细胞毒性,抑制率大于70%;选用0、4、8、16、32μmol/L丹参酮ⅡA作用HUVEC48 h,流式细胞仪检测结果显示:各组接受绿色荧光Calcein-AM的细胞数量与总细胞数比值明显升高,其中8、16、32μmol/L组可显著提高GJIC功能(P<0.01)。q RT-PCR分析结果显示:与空白对照组比较,8、16、32μmol/L丹参酮ⅡA组可显著提高Cx37、Cx40 m RNA表达(P<0.05或P<0.01),并有一定的浓度依赖关系。【结论】丹参酮ⅡA能够提高Cx37、Cx40 m RNA表达,这可能是丹参酮ⅡA增强上皮细胞GJIC功能的机制之一。

Objective To study the effects of tanshinone ⅡA（TⅡA）on the gap junction intercellular communication（GJIC）function of human umbilical vein epithelial cells（HUVEC）and to explore its possible mechanism. Methods Inhibitive effect of TⅡA on the growth of HUVEC was examined by methyl thiazolyl tetrazolium（MTT）assay,the gap junction function was analyzed by fluorescent tracer and flow cytometer under fluorescence microscope.The expression of connexins Cx37 and Cx40 was detected by quantitative real-time polymerase chain reaction（q RT-PCR）. All-trans-retinoic acid（ATRA）served as the positive control drug. Results Treatment with TⅡA for 24, 48 h had an effect on the survival of HUVEC cells, and the half inhibitive concentration（IC50） was15,8.5 μmol/L respectively. Treatment with 32, 64 μmol/L of T Ⅱ A for 48 h had an obvious toxic effcit on HUVEC cells,and the inhibition rate was over 70%. After treatment with 0,4,8,16,32,64 μmol/L of TⅡ A for 48 h, the ratio of Calcein-labeled cells to the total cells showed by flow cytometry was increased obviously,and 8,16,32 μmol/L of T ⅡA markedly promoted GJIC function of HUVEC cells（P〈0.01）. The results of q RT-PCR showed that treatment with 8,16,32 μmol/L of TⅡA could dose-dependently increase the expression levels of Cx37 and Cx40 m RNA（P〈0.05 or P〈0.01 compared with the blank control group）.Conclusion Tanshinone Ⅱ A could competently promote the expression of Cx37 and Cx40, which probably contributes to one of its mechanisms in enhancing the GJIC function of the endothelial cells.