藤黄酸通过阻抑表皮生长因子受体酪氨酸磷酸化抑制大鼠主动脉平滑肌细胞增殖

Gambogic Acid Inhibits Cell Proliferation via Suppressing Epithelial Growth Factor Receptor Tyrosine Phosphorylation in Rat Aortic Smooth Muscle Cell

目的探讨藤黄酸对表皮生长因子诱导大鼠主动脉平滑肌细胞增殖的作用及其机制。方法 CCK8法和3H-胸腺嘧啶掺入法检测不同浓度藤黄酸(0.25、0.5、1.0及2.0μmol/L)对表皮生长因子诱导的血管平滑肌细胞增殖的影响,流式细胞仪检测不同浓度藤黄酸对表皮生长因子诱导的细胞周期转换的影响,Western blotting检测不同浓度藤黄酸对表皮生长因子诱导的表皮生长因子受体和酪氨酸磷酸化的作用,斑点结合检测藤黄酸是否在体外表皮生长因子结合进而影响其活性。结果藤黄酸抑制表皮生长因子诱导的血管平滑肌细胞增殖和DNA合成,藤黄酸抑制表皮生长因子诱导的血管平滑肌细胞周期的演进,藤黄酸抑制表皮生长因子受体和酪氨酸磷酸化,表皮生长因子在体外并不与藤黄酸结合。结论藤黄酸通过对表皮生长因子受体磷酸化的抑制进而抑制表皮生长因子诱导的血管平滑肌细胞增殖,这种抑制可能是通过直接抑制酪氨酸磷酸化引起的,而不是通过藤黄酸与表皮生长因子受体结合引起的。

Aim To investigate the effects of gambogic acid（GA）on rat vascular smooth muscle cell（VSMC） proliferation and migration stimulated by epithelial growth factor（EGF） and its underlying molecular mechanism. Methods The inhibitory effect of GA at various concentration on the proliferation induced by EGF was measured by using CCK8 assay and 3H-thymidine incorporation.The effects of GA on the cell cycle progression stimulated by EGF were analyzed by flow cytometry.The effect of GA EGF receptor（EGFR） and pi-Tyrosine was measured by Western blotting.The capacity of GA binding with EGF was also measured. Results GA inhibited EGF-induced 3H-thymidine incorporation into DNA and proliferation on VSMC.The cell cycle progression was blocked significantly in the GA pretreated cells.GA significantly inhibited expression of pi-EGFR,pi-Tyrosine stimulated by EGF compared with the control group without GA.Dot binding assay shown that a negative binding signal was detected on the GA and BSA spot incubated with EGF. Conclusions These observations show that inhibition effects on VSMC proliferation of GA are mediated by the inhibition of EGFR tyrosine phosphrylation.The inhibitory effects of GA are due to directly tyrosine phosphorylation inhibition,rather than binding with EGF.