青天葵甲醇提取物通过ERK通路抑制人肝癌细胞增殖并诱导其凋亡

目的 研究青天葵甲醇提取物（Nervilia fordii methanol extracts，NFME）体外对人肝癌SMMC7721和HepG2细胞的凋亡作用及其作用机制。方法 紫外-可见分光光度法测定青天葵中总黄酮和总多酚的含量；MTT法检测不同浓度（0，0.25，0.5，1，1.5，2 mg·mL^-1）NFME处理24 h对SMMC7721、HepG2和LO2细胞生长抑制率的影响；克隆形成试验观察NFME对SMMC7721和HepG2细胞克隆形成率的影响；Hoechst凋亡染色观察NFME对SMMC7721细胞凋亡的影响；流式细胞术检测NFME对SMMC7721细胞凋亡和细胞周期的影响；Western blotting测试NFME作用下caspase3、PARP、ERK1/2和c-Raf蛋白磷酸化水平的变化。结果 NFME中总多酚含量为（4.25±0.46）mg·g^-1，总黄酮含量为（4.72±0.13）mg·g^-1；MTT试验结果显示，与对照组相比，在给药24 h时0.5 mg·mL^-1的NFME就能抑制SMMC7721和HepG2细胞的增殖（P<0.001）；克隆形成试验表明，0.125 mg·mL^-1的NFME能够抑制SMMC7721和HepG2细胞集落的形成（P<0.001）；Hoechst凋亡染色观察到0.25 mg·mL^-1的NFME作用24 h能够观察到SMMC7721细胞发生凋亡（P<0.05）；流式细胞试验结果证实0.5 mg·mL^-1的NFME能够诱导SMMC7721细胞的凋亡（P<0.01），凋亡率14.43%，阻滞细胞周期于S期；Western blotting结果表明NFME能使SMMC7721细胞中caspase3发生剪切，随着给药浓度增加其剪切作用越明显（P<0.05），活化的caspase3剪切下游PARP的表达；同时NFME能够降低SMMC7721细胞中ERK1/2和c-Raf蛋白的磷酸化水平（P<0.05）。结论 NFME能抑制人肝癌SMMC7721和HepG2细胞的活性并诱导其发生凋亡，其作用机制可能与抑制ERK信号通路从而诱导细胞凋亡有关。

Nervilia Fordii Methanol Extracts Inhibits Proliferation and Induces Apoptosis of Human Hepatic Carcinoma Through ERK Signaling Pathway

OBJECTIVE To investigate the apoptosis effect and mechanism of Nervilia fordii methanol extracts(NFME) on human hepatic carcinoma SMMC7721 and HepG2 cells in vitro. METHODS UV-visible spectrophotometric method was used to determine total flavonoids and total polyphenols in NFME. The growth inhibition rate of SMMC7721, HepG2 and LO2 treated with different concentrations(0, 0.25, 0.5, 1, 1.5, 2 mg·mL^-1) of NFME for 24 h were detected by MTT. The effect of NFME on the colony formation rate of SMMC7721 and HepG2 cells was observed by clonal test. The effect of NFME on SMMC7721 cell apoptosis was observed by Hoechst apoptosis staining. The effect of NFME on SMMC7721 cell apoptosis and cell cycle were detected by flow cytometry. The phosphorylation level expression of caspase3 and ERK1/2 and c-Raf protein were detected by Western blotting. RESULTS The total polyphenol content in NFME was (4.25±0.46)mg·g^-1, and the total flavonoid content was (4.72±0.13)mg·g^-1. The results of MTT experiments showed that compared with the control group, 0.5 mg·mL^-1 NFME could inhibit the proliferation of SMMC7721 and HepG2 cells(P<0.001); the experiment of clonal formation ability showed that 0.125 mg·mL^-1 NFME could inhibit the formation of SMMC7721 and HepG2 cell colonies(P<0.001); Hoechst apoptosis staining observed that 0.25 mg·mL^-1 NFME induced apoptosis of SMMC7721 cells for 24 h(P<0.05); the flow cytometry results confirmed that 0.5 mg·mL^-1 NFME could induce apoptosis of SMMC7721 cells(P<0.01), and apoptosis rate reached 14.43%, blocked cell cycle in S phase; Western blotting results showed that NFME could make caspase3 in SMMC7721 cells undergo shearing, the shear effect became more pronounced with concentration increasing(P<0.05), and activated caspase3 shears downstream PARP expression; meanwhile, NFME could reduce the phosphorylation level of ERK1/2 and c-Raf proteins in SMMC7721 cells(P<0.05). CONCLUSION The NFME can inhibit the activity of human hepatocellular carcinoma SMMC7721 and HepG2 cells and induces apoptosis The mechanism of induces apoptosis may be related to the inhibition of ERK signaling pathway.