Effects of Notoginsenoside R1 on Characteristics of Esophageal Squamous Cell Carcinoma KYSE150 Cells
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摘要:
目的 探讨源自传统中药三七的活性单体—三七皂苷R1（Notoginsenoside R1，NGR1）对食管鳞癌KYSE150细胞增殖、迁移、侵袭、细胞周期分布及凋亡的影响，并结合生物信息学方法初步预测其潜在作用靶点。 方法 将体外培养的KYSE150细胞分为对照组（含等体积DMSO的完全培养基）、低剂量组（5.7 μg/mL NGR1）、中剂量组（28.8 μg/mL NGR1）和高剂量组（144.0 μg/mL NGR1）。采用CCK-8法检测细胞增殖活力；采用细胞划痕愈合实验及Transwell实验分别评价细胞迁移与侵袭能力；采用流式细胞术检测细胞周期分布及凋亡率；借助网络药理学和分子对接技术预测NGR1抗食管癌的潜在核心靶点。 结果 与对照组相比，NGR1可显著抑制KYSE150细胞增殖，且呈剂量依赖性（P < 0.05）；中、高剂量组划痕愈合率及Transwell穿膜细胞数显著降低、细胞凋亡率显著升高（P < 0.05）；各剂量组G0/G1期细胞比例均显著升高，细胞周期阻滞于G0/G1期（P < 0.05）。网络药理学筛选获得STAT3、CDK1、MMPs（MMP2/7/9）、CASP3等核心靶点，分子对接结果显示NGR1与上述靶蛋白均具有良好的结合活性。 结论 NGR1 能够有效抑制食管癌 KYSE150 细胞的增殖能力，同时显著阻碍该细胞的迁移与侵袭过程，诱导细胞发生凋亡，阻滞细胞周期停滞于 G0/G1 期，其机制可能与多靶点协同调控有关。 Abstract:Objective To investigate the effects of Notoginsenoside R1 （NGR1）, an active monomer derived from the traditional Chinese medicine Panax notoginseng, on the proliferation, migration, invasion, cell cycle distribution, and apoptosis of esophageal squamous cell carcinoma （ESCC） KYSE150 cells, and to preliminarily predict its potential targets using bioinformatics mathods. Methods KYSE150 cells cultured in vitro were divided into a control group （complete medium containing an equal volume of DMSO）, a low-dose group （5.7 μg/mL NGR1）, a medium-dose group （28.8 μg/mL NGR1）, and a high-dose group （144.0 μg/mL NGR1）. Cell proliferation activity was detected by the CCK-8 assay; cell migration and invasion abilities were evaluated by wound healing assay and Transwell assay, respectively; cell cycle distribution and apoptosis rate were detected by flow cytometry; and network pharmacology and molecular docking were used to predict the potential core targets of NGR1 against esophageal cancer. Results Compared with the control group, NGR1 significantly inhibited KYSE150 cell proliferation in a dose-dependent manner （P < 0.05）. In the medium- and high-dose groups, the wound healing rate and the number of cells penetrating the Transwell membrane were significantly decreased, while the apoptosis rate was significantly increased （P < 0.05）. The proportion of cells in the G0/G1-phase was significantly increased in all dose groups, indicating cell cycle arrest at G0/G1 phase （P < 0.05）. Network pharmacology screening identified core targets such as STAT3, CDK1, MMPs （MMP2/7/9） and CASP3, and molecular docking results showed that NGR1 had good binding activity with the above target proteins. Conclusion NGR1 can effectively inhibit the proliferative capacity of esophageal cancer KYSE150 cells, significantly impede their migration and invasion, induce apoptosis, and arrest the cell cycle at the G0/G1 phase. Its mechanism may be related to coordinated regulation of multiple targets. -
Key words:
- Esophageal neoplasms /
- Notoginsenoside R1 /
- Cell proliferation /
- Cell migration /
- Bioinformatics
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图 1 NGR1对KYSE150细胞增殖活力的影响
A： NGR1 的剂量-效应（IC50）拟合曲线 ；B：细胞活力随时间（0、24、48、72 h） 变化的折线图 。
Figure 1. Effects of NGR1 on the proliferative viability of KYSE150 cells
图 2 NGR1对KYSE150细胞划痕愈合能力的影响（100×）
A：划痕实验检测不同处理后的KYSE150 细胞迁移能力（标尺100 μm）；B：划痕愈合率（%）随时间变化的折线图。
Figure 2. Effect of NGR1 on the wound healing ability of KYSE150 cells （100×）
图 3 NGR1对KYSE150细胞侵袭能力的影响
A：Transwell 实验检测不同处理后的 KYSE150 细胞侵袭能力（标尺100 μm）；B：侵袭实验统计图；*P < 0.05； **P < 0.01；***P < 0.001; ****P < 0.0001；ns: P ≥ 0.05。
Figure 3. Effect of NGR1 on the invasive ability of KYSE150 cells
图 4 NGR1对KYSE150细胞周期分布的影响
A：流式细胞术检测不同处理后的 KYSE150 细胞周期；B：周期实验统计图；***P < 0.001; ****P < 0.0001。
Figure 4. Effect of NGR1 on cell cycle distribution in KYSE150 cells
图 5 NGR1对KYSE150细胞凋亡的影响
A:Annexin V-FITC/PI检测KYSE150细胞凋亡率；B：KYSE150细胞凋亡率分析；**P < 0.01；****P < 0.0001；ns：P≥0.05 。
Figure 5. Effect of NGR1 on apoptosis of KYSE150 cells
图 6 NGR1与核心靶点蛋白的分子对接结果
A：CDK1；B：STAT3；C：CASP3；D：MMP2。各小图左侧为靶点蛋白的整体三维结构，右侧方框为结合口袋局部放大；位于结合口袋中央、不带残基编号的连续多环彩色棍状结构为NGR1，标注三字母缩写及序号（如GLU162、ARG170、THR142、LYS51等）者为结合口袋内的关键氨基酸残基，黄色虚线表示NGR1与氨基酸残基之间形成的氢键（数字为氢键键长，Å）。
Figure 6. Molecular docking results of NGR1 with core target proteins
表 1 主要试剂
Table 1. Main reagents
试剂 / 耗材名称 生产厂家 三七皂苷 R1 标准品（≥98%（HPLC）） 成都普瑞法科技开发有限公司 DMEM 高糖培养基、胎牛血清（FBS） 武汉普诺赛生命科技有限公司 CCK-8 细胞增殖检测试剂盒、结晶紫染色液 北京索莱宝科技有限公司 Annexin V-FITC/PI 双染凋亡检测试剂盒、细胞周期检测试剂盒、Matrigel 基质胶 美国 BD 公司 Transwell 小室 美国 Corning 公司 二甲基亚砜（DMSO） 美国 Sigma 公司 胰蛋白酶 - EDTA 消化液 上海原液生物科技有限公司 PBS 缓冲液粉剂 北京兰杰柯科技有限公司 
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