Objective: To study the effect and mechanism of angiotensin (Ang II) on the proliferation of human hepatocellular carcinoma HepG2 cells. Methods: The effects of different concentrations of Ang II's (10(-8)-10(-4) mol/L) on proliferated hepatocellular carcinoma HepG2 cells were detected by CCK-8 assay. The expression of angiotensin II type 1 receptor (AT1) protein and activation of ERK1/2 protein in hepatocellular carcinoma HepG2 cells after processing with Ang II were assayed by Western blot. The cells were pretreated with candesartan (AT1 receptor antagonist), sorafenib (Raf kinase inhibitor) and PD98059 (ERK1/2 inhibitor) for 1.5 h and then Ang II (10(-6) mol/L) was added. CCK-8 assay was used to determine whether it could reverse the proliferation of Ang II, and ERK phosphorylation levels were detected by Western blot. The changes in Bcl-2 and c-myc gene expression before and after Ang II processing were detected by Rt-PCR. According to different data, t-test, one-way analysis of variance or SNK method were used for statistical analysis. Results: HepG2 cells treated with different concentrations of Ang II promoted cell proliferation after 24h and 48h. After 24 h, cell vitality was strongest with Ang II concentration 10(-5) mol/L and the absorbance value was 0.990 8±0.097 8; and again after 48 h, the cell viability was strongest with Ang II concentration 10(-6) mol/L and the absorbance value was 1.302 7 ± 0.030 9. Moreover, the pro-proliferation effect of Ang II on HepG2 cells blocked candesartan, sorafenib and ERK1/2 isolated inhibitors. After treatment with 10(-6) mol/L Ang II, Western blot showed that Ang II significantly promoted AT1 receptor expression and phosphorylation of ERK1/2 protein confirmed that Ang II activated the AT1/RAF/ERK1/2 signaling pathway. In addition, Rt-PCR detection showed that the downstream of Bcl-2 and c-myc genes expressions rose significantly when the concentration of Ang II ranged from 10(-8) to 10(-6) mol/L. Conclusion: Ang II can promote the proliferation of HepG2 cells by activating AT1/Raf /ERK1/2 signaling pathway and enhance the downstream of Bcl-2 and c-myc gene expression.
目的： 研究血管紧张素II（Ang II）对人肝癌细胞系HepG2细胞增殖能力的影响及其机制。 方法： 通过CCK-8法检测不同浓度Ang II（10(-8)～10(-4) mol/L）处理肝癌细胞系HepG2后对细胞增殖的影响。采用Western blot法检测Ang II处理后HepG2细胞内血管紧张素1型受体（AT1）蛋白表达和细胞外信号调节激酶（ERK）1/2蛋白活化水平。再分别用坎地沙坦（AT1受体拮抗剂）、索拉菲尼（Raf激酶抑制剂）和PD98059（ERK1/2抑制剂）预处理细胞1.5 h后加入Ang II（10(-6) mol/L），CCK-8法检测其能否逆转Ang II的促增殖作用，并用Western blot检测其ERK磷酸化水平。用Rt-PCR法检测Ang II处理前后Bcl-2和c-myc基因表达水平的变化。据资料不同分别采用t检验、单因素方差分析或SNK法进行统计学分析。 结果： 不同浓度Ang II处理HepG2细胞24 h和48 h后均可促进细胞增殖，Ang II浓度为10(-5) mol/L时24 h后细胞活力最强，吸光度值为0.990 8±0.097 8；Ang II浓度为10(-6) mol/L时48 h后细胞活力最强，吸光度值为1.302 7±0.030 9。且Ang II对细胞的促增殖作用可被坎地沙坦、索拉菲尼和ERK1/2抑制剂单独阻断。用10(-6) mol/L的Ang II处理细胞后通过Western blot检测发现Ang II可显著促进AT1受体的表达和ERK1/2蛋白的磷酸化，证明Ang II可激活AT1/Raf/ERK1/2信号通路。此外，通过Rt-PCR检测发现当Ang II浓度为10(-8)～10(-6) mol/L时可显著提高下游Bcl-2和c-myc基因的表达。 结论： Ang II可通过激活AT1/Raf/ERK1/2信号通路促进HepG2细胞增殖，并可提高下游Bcl-2和c-myc基因的表达。.
Keywords: Angiotensin II; Angiotensin type 1 receptor; Carcinoma, hepatocellular; Extrallular signal-regulated kinase 1/2; Raf.