冬凌草甲素抑制HCT116细胞增殖与p38 MAPK信号通路相关性研究

Relationship between anti-proliferation effect of Oridonin on HCT116 cells and p38 MAPK signal pathway

目的研究冬凌草甲素(ORI)对人源结肠癌细胞HCT116的增殖抑制作用,并探讨其可能的分子机制。方法采用结晶紫染色研究ORI(5、10、15、20、25μmol/L)对HCT116细胞的增殖抑制作用;采用Western blotting技术研究ORI(10、15、20μmol/L)对HCT116细胞中增殖细胞核抗原(PCNA)、凋亡相关指标(Caspase-3和Cleaved-caspase-3)、p38 MAPK信号通路相关指标(Smad1/5/8、p-Smad1/5/8、p38、p-p38)蛋白表达的影响;采用Western blotting、凝胶电泳技术分析ORI对BMP7蛋白、mRNA表达的影响;转染重组BMP7腺病毒(Ad BMP7)实现BMP7的外源性过表达,应用BMP7抗体(anti-BMP7)降低BMP7水平,检测ORI是否通过调节BMP7表达影响HCT116细胞增殖凋亡、p38 MAPK信号通路。结果与对照组比较,ORI明显抑制HCT116细胞增殖,下调PCNA蛋白水平;明显上调Caspase-3、Cleaved caspase-3、p-p38蛋白表达,且呈浓度与时间相关性,对Smad1/5/8、P-Smad1/5/8蛋白表达无明显影响;明显上调BMP7蛋白及RNA表达;外源性过表达BMP7加强了ORI对上述蛋白表达的调控,anti-BMP7则部分抑制了ORI的作用。结论 ORI抑制HCT116细胞的增殖,其机制可能与上调BMP7蛋白表达进而激活p38 MAPK信号通路相关。

Objective To investigate the proliferation inhibition effect and the possible mechanism of Oridonin（ORI）on human colon cancer HCT116 cells.Methods The inhibitory effects of ORI（5,10,15,20,25 μmol/L） on the proliferation of HCT116 cells were tested by crystal violet staining.The effects of ORI（15,20,25 μmol/L） on the expression of proliferating cell nuclear antigen（PCNA）,apoptosis related index（Caspase-3 and Cleaved-caspase-3） and p38 MAPK signal pathway related protein（Smad1/5/8,p-Smad1/5/8,p38 and p-p38） in HCT116 cells were studied by Western blotting assay.The effect of ORI on the expression of BMP7 protein and m RNA was analyzed by Western blotting and gel electrophoresis.Next,the exogenous overexpression of BMP7 were realized by transfecting recombinant adenovirus（Ad BMP7）,and the BMP7 antibody（anti-BMP7）,which could reduce the expression of BMP7,was used to detect the influence of ORI on proliferation and apoptosis of HCT116 cells and p38 MAPK signal pathway by regulating the expression of BMP7.Results Compared with control group,ORI could inhibit the proliferation of HCT116 cells obviously,down-regulated the PCNA protein level; Western blotting results showed that ORI up-regulated the protein level of BMP7,Caspaes-3,Cleaved caspase-3 and p-p38 in a time and concentration dependent manner,with no obvious effect on the expression of Smad1/5/8 and P-Smad1/5/8 protein and obvious up-regulative effect on the BMP7 protein and m RNA.Over expression of BMP7 strengthened the regulative level of above-mentioned proteins,while anti-BMP7 partly inhibited such effects of ORI.Conclusion ORI can inhibit the proliferation of HCT116 cells,which may be related to the up-regulation of BMP7 protein expression with view to activating of the p38 MAPK signal pathway.