党参多糖通过PI3K/AKT信号通路抑制瘢痕疙瘩成纤维细胞增殖和诱导细胞周期阻滞的实验研究

Codonopsis pilosula polysaccharide inhibits the proliferation of keloid fibroblasts and induces cell cycle arrest through PI3K/AKT signaling pathway:an experimental study

目的:探讨党参多糖对人瘢痕疙瘩成纤维细胞增殖和细胞周期进程的影响及其分子机制。方法:四唑盐比色测定法[3-(4,5-Dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT]检测党参多糖对细胞存活率的影响,筛选党参多糖加药浓度,采用平板克隆实验检测瘢痕疙瘩成纤维细胞的克隆形成能力,流式细胞术检测细胞周期比例和细胞凋亡,蛋白免疫印迹法(Western blot)分析增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、细胞周期蛋白D1(Cyclin D1)、Ⅰ型胶原蛋白(CollagenⅠ)、Ⅲ型胶原蛋白(CollagenⅢ)、磷脂酰肌醇-3激酶(phosphoinositide 3-kinase,PI3K)和丝苏氨酸蛋白激酶(protein kinase B,AKT)蛋白磷酸化水平。结果:当党参多糖浓度小于80μmol/L时对瘢痕疙瘩成纤维细胞无明显毒性作用,本实验选择浓度为10、20、40μmol/L的党参多糖作为后续实验加药标准浓度;与0μmol/L组相比,10、20和40μmol/L组瘢痕疙瘩成纤维细胞克隆形成能力降低,细胞周期阻滞于G_(0)/G_(1)期,PCNA、Cyclin D1、CollagenⅠ和CollagenⅢ的表达下调,细胞凋亡率升高,PI3K和AKT磷酸化水平降低(P<0.05)。结论:党参多糖能够抑制瘢痕疙瘩成纤维细胞增殖和细胞周期进程,其分子机制可能与抑制PI3K/AKT信号通路的激活有关。

Objective:To investigate the effects of Codonopsis pilosula polysaccharide on the proliferation and cell cycle progression of human keloid fibroblasts,and the possible molecular mechanisms.Methods:The 3-(4,5-Dimethylthazol-2-yl)-2,5-diphenyltetrazo⁃lium bromide assay was used to determine the effect of C.pilosula polysaccharide on cell survival rate and select the concentration of C.pilosula polysaccharide.The clone-forming ability of keloid fibroblasts was determined by plate cloning experiment.The cell cycle distribution and apoptosis were determined by flow cytometry.Western blot was used to analyze proliferating cell nuclear antigen,cy⁃clin D1,collagen I,collagenⅢ,and the phosphorylation levels of phosphatidylinositol 3-kinase and protein kinase B.Results:When the concentration of C.pilosula polysaccharide was less than 80μmol/L,there was no obvious toxic effect on keloid fibroblasts.In this experiment,C.pilosula polysaccharide was added at the concentrations of 10,20,and 40μmol/L.Compared with the 0μmol/L group,the clone-forming ability of keloid fibroblasts decreased in the 10,20,and 40μmol/L groups;cell cycle was arrested at the G_(0)/G_(1) phase;the expression of proliferating cell nuclear antigen,cyclin D1,collagenⅠ,and collagenⅢwas down-regulated;cell apoptosis was increased;the phosphorylation levels of PI3K and AKT decreased(P<0.05).Conclusion:Codonopsis pilosula polysaccharide can inhibit the proliferation and cell cycle progression of keloid fibro⁃blasts,and the molecular mechanisms may be related to the inhibi⁃tion of the PI3K/AKT signal pathway.