人参皂苷单体Rh_2抑制小鼠前胃癌系细胞增殖及其机制

Mechanism of anti-proliferation effect of ginseng Rh_2(G-Rh_2) on mouse MFC gastric cells

目的探讨人参皂苷Rh2(G-Rh2)对小鼠前胃癌系(MFC)细胞增殖的抑制作用及其机制。方法分别对MFC正常细胞组和G-Rh2(3、10、30mg.L-1)组经MTT法检测MFC细胞活性;倒置显微镜和Hoechst33258荧光染色观察凋亡细胞形态;AnnexinV-FITC双染法分析G-Rh2对细胞凋亡率的影响;分别对MFC正常细胞组、G-Rh2(10mg.L-1)处理不同时间(30min、1、2、4h)组、SP600125(5μmol.L-1)预处理2h+G-Rh2(10mg.L-1)不同时间(30min、1、2、4h)给药组经MTT法检测MFC细胞活性;Westernblot法检测G-Rh2(10mg.L-1)处理不同时间(5、15、30、45min、1、2、4h)组p-JNK(c-JunN-terminalkinase1)激酶和p-c-jun在MFC细胞中的活性,免疫细胞化学染色法检测caspase-3阳性细胞的表达率。结果G-Rh2对无血清MFC细胞有明显细胞毒活性,呈时间和剂量依赖关系;能明显诱导细胞皱缩,核染色质固缩,核碎裂,形成大约为180～200bp或其多聚体组成的寡核苷酸片断,凋亡细胞比率上升。G-Rh2处理后4h内,p-JNK激酶和p-c-jun活性持续升高,此过程可被预处理2h的SP600125(5μmol.L-1)部分抑制。结论人参皂苷G-Rh2可诱导MFC细胞凋亡,其作用机制之一可能是通过激活JNK信号传导途径,并最终增加caspase-3的激活而完成。

Aim To investigate the molecular mechanism of anti-proliferation effect of Ginsenoside Rh2 （ G-Rh2 ） on mouse MFC gastric cells. Methods The cell viability was determined by MTT method and the state of cell proliferation was analyzed. The morphologic changes were observed by invert microscope and fluo- rescence microscope （Hoechst 33258）. Early apoptotic rate was detected by Annexin V-FITC through flow cytometry. Western blot was used to detect the p-JNK and p-c-jun activity in MFC cells. Immunocytochemistry staining was used to detect caspase-3 positive cells. Results G-Rh2 could inhibit proliferation of MFC cells in a dose-and-time-dependent manner. The morphological changes of apoptosis were observed in MFC cells after G-Rh2 treatment. The apoptotic ratio was in- creased. The phosphorylation of c-Jun and c-Jun NH2- terminal kinase（JNK） activity increased with time within 4 h compared with that of control. The expression of caspase-3 positive cells was increased. But all the above could be partly inhibited by pre-treatment with SP600125 （5 μmol·L^-1） for 2 h. Conclusion GRh2 can decrease cell viability by inducing apoptosis and the process may be associated with the activation of JNK transduction pathway and expression of caspase-3.