丹酚酸B对鼻息肉成纤维细胞的增殖抑制及诱导凋亡的研究

Effect of salvianolic acid B on proliferation and apoptosis of human nasal polyps fibroblasts

目的:探讨丹酚酸B对人鼻息肉成纤维细胞(NPFs)增殖及凋亡的影响及其分子机制。方法:采用5、10、20、40μmol/L不同浓度丹酚酸B处理体外培养的鼻息肉来源的NPFs,CCK-8法确定药物丹酚酸B最佳作用浓度,将实验分为对照组、TGF-β1刺激组、丹酚酸B组、TGF-β1和丹酚酸B联合处理组。MTT法检测各组细胞增殖的情况,Annexin V-FITC/PI双标法检测细胞凋亡率,Western Blot法检测凋亡相关蛋白Bcl-2、Bax表达,分光光度法检测Caspase-3活性。结果:CCK-8结果显示丹酚酸B对NPFs增殖具有抑制作用,抑制率与丹酚酸B浓度呈正相关(P<0.05),选取丹酚酸B浓度为10μmol/L进行后续实验,MTT结果显示丹酚酸B能抑制TGF-β1对NPFs的促增殖作用(P<0.01);Annexin V-FITC/PI双标法结果显示丹酚酸B促进NPFs凋亡(P<0.05);经丹酚酸B作用后,Caspase-3的活性升高(P<0.05);Western Blot结果显示丹酚酸B能使Bcl-2蛋白表达下降,Bax蛋白表达升高,Bax/Bcl-2比值增大,差异有统计学意义(P<0.05)。结论:丹酚酸B对NPFs有抑制增殖和诱导凋亡的作用,其机制可能与下调Bcl-2蛋白、上调Bax蛋白表达、升高Caspase-3活性有关。

Objective：The aim of this study is to discuss the effects and mechanism of salvianolic acid B（SAB）on the proliferation and apoptosis of human nasal polyps fibroblasts（NPFs）.Method：The NPFs were treated with SAB at different concentrations（5,10,20,40μmol/L）.CCK-8 assay was used to determine the inhibition of SAB on the proliferation of NPFs and determined the optimum concentration for the following experiments.The NPFs were divided into four groups：a）control group：the cells were cultured in DMEM without TGF-β1 or salvianolic acid B;b）the TGF-β1 group：the cells were cultured in DMEM containing 5 ng/ml TGF-β1;c）the SAB group：the cells were cultured in DMEM containing SAB at the optimum concentration by CCK-8 assay;d）the TGF-β1＋SAB treatment groups：the cells were cultured in DMEM containing 5 ng/ml TGF-β1 and SAB at the optimum concentration by CCK-8 assay.MTT assay was used to determine the proliferation of NPFs in four groups.The apoptosis rate was analyzed by Annexin-V FITC/PI double staining.The activities of Caspase-3 were detected with spectrophotometric method.Apoptosis related proteins Bcl-2 and Bax were measured by western blot.Result：CCK-8 assay showed that SAB could inhibite NPFs proliferation in a dose-dependent pattern（P〈0.05）.The following experiments were carried out with the concentration of salvianolic acid B at 10μmol/L.MTT results showed that SAB could inhibit the proliferation of NPFs stimulated by TGF-β1（P〈0.01）.Annexin-V FITC/PI double staining showed that SAB could induce apoptosis of NPFs（P〈0.05）.The activities of Caspase-3 were increased with SAB（P〈0.05）.Western blot results showed that SAB reduced the Bcl-2 protein expression,and increased the Bax protein expression and the ratio of Bax/Bcl-2（P〈0.05）.Conclusion：Salvianolic acid B could inhibit proliferation and induce apoptosis of NPFs.Its mechanism may be associated with increasing the activities of Caspase-3 by inhibiting the expression of Bcl-2 protein and promoting the expression of Bax protein.