基于对癌细胞作用的人参与黄芪的药性异同点的分子机制研究

Molecular mechanism of the similarities and differences in medicinal properties between ginseng and milkvetch root based on their effects on cancer cells

目的比较人参、黄芪对不同癌细胞的作用,探讨二者药性异同的分子机制。方法CCK-8法检测质量浓度梯度的人参标准提取物(RSE75)、黄芪标准提取物(HQE75)对不同癌细胞(人源肝癌细胞Hep G2、鼠源肝癌细胞H22、人源肺癌细胞H1299、人源肺癌细胞A549、人源食管癌细胞EC109及鼠源食管癌细胞AKR)增殖活性的影响;RT⁃qPCR法检测RSE75、HQE75对白细胞介素-6(IL⁃6)、肿瘤坏死因子α(TNF⁃α)、转化生长因子-β(TGF⁃β)mRNA表达及A549细胞中DNA甲基转移酶(DNMTs)/10-11易位甲基胞嘧啶双加氧酶2(TET2)平衡关系的影响;利用克隆形成实验、Annexin V⁃FITC/PI染色技术、流式细胞仪分别检测RSE75、HQE75对A549细胞克隆形成率、凋亡水平、周期变化的影响;转录组测序技术探索RSE75及HQE75治疗肺癌的潜在靶点和作用途径。结果与空白对照组比较,RSE75组癌细胞存活率均降低,多数HQE75组差异无统计学意义;RSE75组及HQE75组IL⁃6、TNF⁃α和TGF⁃βmRNA表达水平均降低;RSE75可抑制A549细胞的克隆形成率,且随剂量增加抑制率降低,而HQE75无明显抑制作用;RSE75及HQE75均可诱导A549细胞凋亡,并将DNA复制阻滞在G1/S期,相同质量浓度下,RSE75的诱导凋亡作用强于HQE75;RSE75组DNMTs mRNA表达水平降低、TET2 mRNA表达水平升高,HQE75组DNMTs、TET2 mRNA表达水平均降低。RNA测序研究结果表明,RSE75与HQE75有着共同作用靶点与作用途径,共同作用靶点主要有白细胞介素-8、趋化因子配体1、细胞间黏附分子-1、趋化因子配体2等,共同作用途径主要有白细胞介素-17、核转录因子-κB、肿瘤坏死因子信号通路等,多为免疫调节途径;不同点在于,人参能够通过血管内皮生长因子A、早期生长应答因子1等靶点作用于缺氧诱导因子-1信号通路等抑制癌细胞增殖,黄芪能够通过IL⁃6、核转录因子等靶点调控氨基酸代谢、核苷酸结合寡聚化结构域样受体信号通路等发挥免疫调节作用。结论人参与黄芪都具有明显的免疫调节作用,同时,人参还具有明显的抑制癌细胞增殖作用;人参能调节DNMTs/TET2平衡,而黄芪主要抑制DNMTs表达。本研究从分子水平上阐明了二者在抗癌作用上药性的不同,为同为补气药的人参和黄芪的临床应用提供了科学依据,也为中药药性对比分子机制研究提供了思路。

Objective We aimed to(i)compare the effects of renshen(ginseng)and huangqi(milkvetch root)on different cancer cells and(ii)elucidate the molecular mechanisms underlying the similarities and differences in their medicinal properties.Methods The effects of ginseng standard extract(RSE75)and milkvetch root standard extract(HQE75)on the proliferative activity of different cancer cells(Hep G2,H22,H1299,A549,EC109,and AKR)were determined by the CCK-8 assay.The effects of RSE75 and HQE75 on the mRNA expression levels of immune factors IL⁃6,TNF⁃α,and TGF⁃βand the balance between DNA methyltransferases(DNMTs)and TET2 in A549 cells were determined by RT⁃qPCR.The effects of RSE75 and HQE75 on the clonal formation rate,apoptosis,and the cell cycle of A549 cells were detected by a clonal formation assay,Annexin V⁃FITC/PI staining,and flow cytometry,respectively.The potential targets and pathways of RSE75 and HQE75 in the treatment of lung cancer were explored by RNA sequencing technology.Results Compared with the blank group,the survival rate of cancer cells in the RSE75 group was decreased,but there was no significant change in the HQE75 group.The mRNA expression levels of IL⁃6,TNF⁃α,and TGF⁃βwere decreased in the RSE75 and HQE75 groups.RSE75 inhibited the clonal formation of A549 cells in a concentration⁃dependent manner,while HQE75 had no obvious inhibitory effect.Both RSE75 and HQE75 significantly induced apoptosis in A549 cells and blocked DNA replication in the G1/S phase.At the same concentration,RSE75 has a stronger effect on inducing apoptosis than HQE75.The mRNA expression level of DNMTs in the RSE75 group was decreased,the mRNA expression level of TET2 in the RSE75 group was increased,and the mRNA expression level of both DNMTs and TET2 was decreased in the HQE75 group.The RNA sequencing result showed that RSE75 and HQE75 had common targets,including CXCL8,CXCL1,ICAM1,and CCL2.The common pathways of action mainly included the IL-17 signaling pathway,the NF⁃κB signaling pathway,and the TNF signaling pathway,and most of them were involved in immune regulation.The main difference is that renshen can inhibit the proliferation of cancer cells through the HIF-1 signaling pathway through VEGFA,EGR1,and other targets,while huangqi can regulate amino acid metabolism and the NOD⁃like receptor signaling pathway through IL⁃6,TNF,and other targets.Conclusion Studies on the anti⁃cancer effects and the underlying molecular mechanisms have shown that renshen and huangqi have obvious immunomodulatory effects,but renshen also had significant inhibitory effects on cancer cell proliferation.Renshen could regulate the DNMTs/TET2 balance,while huangqi could inhibit the expression of DNMTs.This study elucidated the different properties of renshen and huangqi the molecular level and provides not only a scientific basis for clinical application of renshen and huangqi,which are both qi⁃tonifying medicinals,but also ideas for the study of the molecular mechanisms underlying the effects of other TCMs.