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[摘要]
目的 探讨重楼总皂苷(Rhizoma Paridis total saponin,RPTS)在体外对人胃癌MKN-45细胞增殖、迁移和侵袭能力的影响,探讨其作用的可能机制。方法 体外培养MKN-45细胞,取对数生长期细胞,加入不同质量浓度RPTS(2.5、5.0、10.0、20.0、40.0 μg/mL)处理24 h,采用MTT法检测细胞增殖情况;细胞划痕实验、Transwell小室实验检测RPTS对MKN-45细胞迁移和侵袭的影响;酶联免疫吸附实验(ELISA)检测RPTS对LiCl诱导的MKN-45细胞上清液中基质金属蛋白酶-9(MMP-9)的上调表达的影响;免疫印迹技术(Western blotting)和实时荧光定量PCR(qRT-PCR)技术分别检测RPTS(10、20、40 μg/mL)对LiCl诱导的MKN-45细胞中肿瘤侵袭转移相关分子血管内皮生长因子(VEGF)、环加氧酶-2(COX-2)以及LiCl作用靶点糖原合酶激酶-3β(GSK-3β)蛋白和基因表达水平的影响。结果 与对照组比较,RPTS 5.0、10.0、20.0、40.0 μg/mL可明显下调MKN-45细胞的增殖活性(P<0.05、0.001);RPTS 2.5、5.0、10.0μg/mL可抑制MKN-45细胞的迁移和侵袭能力(P<0.01、0.001)。与模型组比较,RPTS能够显著下调LiCl诱导的MKN-45细胞上清液中MMP-9的表达水平(P<0.05、0.01);下调细胞中VEGF、COX-2 mRNA和蛋白表达水平;上调LiCl作用位点GSK-3β mRNA和蛋白表达水平(P<0.05、0.001)。结论 RPTS具有体外抑制MKN-45细胞迁移和侵袭的能力,其作用机制可能与调控Wnt/β-catenin信号通路有关。
[Key word]
[Abstract]
Objective To investigate the effect and the possible mechanism of Rhizoma Paridis total saponin (RPTS) on human gastric cancer cell line MKN-45 proliferation, migration and invasion in vitro. Methods MKN-45 cells were cultured in vitro and treated respectively with indicated concentrations of RPTS (2.5, 5.0, 10.0, 20.0, and 40.0 μg/mL) for 24 h, and cell viability of cell proliferation was detected by MTT assay; The invasive and metastatic ability of MKN-45 treated with indicated concentrations of RPTS (2.5, 5.0, 10.0 μg/mL) was detected by Transwell migration assay and wound healing assay; Elisa assay was employed to detect the concentrations of MMP-9 induced by LiCl after RPTS administration (10, 20, and 40 μg/mL) in the cell supernatant; Western blotting and qRT-PCR were respectively performed to investigate the invasion and migration related protein and mRNA level of VEGF, COX-2, and GSK-3β in RPTS-treated MKN-45 after LiCl stimulation for 24 h. Results Compared with the control group, RPTS (10, 20, and 40 μg/mL) significantly inhibited the proliferation of MKN-45 cells (P<0.05 and P<0.001); RPTS (2.5, 5.0, 10.0 μg/mL) suppressed the invasion and migration of MKN-45 cells (P<0.05 and P<0.001); Compared with the model group, RPTS significantly downregulated the expression of MMP-9 in the cell supernatant of MKN-45 cells induced by LiCl (P<0.05 and P<0.01), and RPTS also decreased the protein and mRNA expression level of VEGF and COX-2, but it significantly upregulated the expression of GSK-3β at the protein and mRNA level (P<0.05 and P<0.001). Conclusion RPTS play a pivotal role in suppressing the invasion and migration of MKN-45 cells in vitro, and its mechanism may be related to the regulating effects of the Wnt/β-catenin pathway in the human gastric adenocarcinoma cell.
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[基金项目]
国家自然科学基金面上项目(81573864);安徽中医药大学校级科研自然重点项目(2019zrzd03);安徽中医药大学校级科研自然面上项目(2016zr004);安徽中医药大学校级科研自然面上项目(2016zr001)