莪术二酮通过VEGF/VEGFR2信号通路对肝癌HepG2细胞微环境下HHSEC增殖的影响

曹如柔 周坚 王其美 章茜 王容容 陈茂

〔摘要〕 目的 探讨莪术二酮对肝癌HepG2细胞微环境下的人肝窦内皮细胞（human hepatic sinusoidal endothelial cells， HHSEC）增殖的抑制作用，并阐明其可能机制。方法 CCK-8法检测不同浓度（0.5%、1%、2%、4%、8%）的HepG2细胞上清液处理HHSEC后的细胞增殖率;HHSEC分为空白组、阳性对照组及莪术二酮低、中、高剂量组，分别予以M199培养基、含1 μg/L顺铂注射液的M199培养基、0.5、1、2 μg/L浓度的莪术二酮工作液培养。应用RT-PCR法和Western blot法检测不同浓度莪术二酮干预后HHSEC内的VEGF、VEGFR2 mRNA及蛋白表达情况。结果 2%、4%及8%的HepG2细胞上清液可促进HHSEC的增殖（P<0.05）;在4% HepG2细胞上清液作用下，0.5、1、2 μg/L莪术二酮均可抑制HepG2细胞上清液对HHSEC的增殖作用（P<0.05），随着浓度的增加，HHSEC增殖率降低明显，不同浓度组间差异均有统计学意义（P<0.05）。在HepG2细胞微环境下，与空白组相比，莪术二酮高剂量组HHSEC中VEGF mRNA和蛋白表达明顯降低（P<0.01），莪术二酮中、高剂量组VEGFR2 mRNA和蛋白的表达均明显降低（P<0.01）。结论 莪术二酮对HepG2细胞微环境下的HHSEC具有抗增殖活性，其作用机制可能与抑制HepG2细胞微环境下HHSEC中VEGF和VEGFR2的表达有关。

〔关键词〕 肝癌;莪术二酮;VEGF/VEGFR2;HepG2细胞;人肝窦内皮细胞

〔中图分类号〕R285.5       〔文献标志码〕A        〔文章编号〕doi：10.3969/j.issn.1674-070X.2021.12.004

The Effect of the Curdione on the Proliferation of HHSEC Under the Microenvironment of

HepG2 Cells via VEGF/VEGFR2 Signaling Pathway

CAO Rurou1， ZHOU Jian2*， WANG Qimei2， ZHANG Xi2， WANG Rongrong2， CHEN Mao2

（1. Hunan University of Chinese Medicine， Changsha， Hunan 410208， China; 2. Department of Oncology，

The Affiliated Hospital of Hunan Academy of Chinese Medicine， Changsha， Hunan 410006， China）

〔Abstract〕 Objective To explore the inhibitory effect of curdione on the proliferation of human hepatic sinusoidal endothelial cells （HHSEC） in the microenvironment of liver cancer HepG2 cells， and to clarify its possible mechanism. Methods CCK-8 method was used to detect the cell proliferation rate of HepG2 cells supernatants of different concentrations （0.5%， 1%， 2%， 4%， 8%） after HHSEC treatment; HHSEC was divided into blank group， positive control group and curdione low， medium， and high-dose groups， and were cultured with M199 medium， M199 medium containing 1 μg/L cisplatin injection， and working solution of curdione at 0.5， 1， and 2 μg/L. RT-PCR and Western blot were used to detect the expression of VEGF and VEGFR2 mRNA and protein in HHSEC after the intervention of different concentrations of curdione. Results 2%， 4% and 8% of HepG2 cells supernatants can promote the proliferation of HHSEC （P<0.05）; under the action of 4% HepG2 cells supernatants， 0.5， 1， 2 μg/L curdione can inhibit the proliferation effect of HepG2 cells supernatant on HHSEC （P<0.05）. With the increase of concentration， the proliferation rate of HHSEC decreased significantly， and the differences between groups of different concentrations were statistically significant （P<0.05）. In the microenvironment of HepG2 cells， compared with the blank group， the expression of VEGF mRNA and protein in HHSEC of the curdione high-dose group was significantly reduced （P<0.01）， and the expression of VEGFR2 mRNA and protein in curdione middle and high-dose group were both decreased （P<0.01）. Conclusion Curdione has anti-proliferative activity on HHSEC in HepG2 cells microenvironment， and its mechanism of action may be related to inhibiting the expression of VEGF and VEGFR2 in HHSEC in HepG2 cells microenvironment.

〔Keywords〕 hepatic carcinoma; curdione; VEGF/VEGFR2; HepG2 cells; human hepatic sinusoidal endothelial cells

原发性肝癌是一种常见的恶性消化道肿瘤，我国每年约有40万以上的原发性肝癌患者，占世界肝癌总人数的一半以上[1-2]。莪术二酮作为莪术中含量最高的倍半萜类物质，其药理作用广泛，具有抗血小板聚集、减轻化疗不良反应等活性，还能降低人乳腺癌HCC1937细胞的迁移和侵袭能力[3-5]，但其抗癌机制尚未明确。血管内皮生长因子（vascular endothelial growth factor， VEGF）是促进血管生成的调节因子[6]，其高表达对诱导肿瘤血管生成，加速肝癌的发生、增殖、转移起重要作用[7-8]。……