穿心莲内酯纳米粒对口腔鳞癌HN6细胞株和白斑Leuk1细胞株抑制作用的实验研究

doi:10.3969/j.issn.1005-4979.2020.04.005

摘要/Abstract

摘要：

目的：研究穿心莲内酯（andrographolide, AND）纳米化（穿心莲内酯纳米粒，andrographolide solid lipid nanoparticles, AND-SLNs）后，对口腔鳞癌的HN6细胞株和口腔白斑的Leuk1细胞株抑制作用的变化。方法：利用高效液相色谱（high performance liquid chromatography，HPLC）法、四氮唑蓝盐化合物细胞增殖实验（MTS法）、流式细胞仪，对比分析AND-SLNs和AND对HN6细胞、Leuk1细胞摄取药物能力和细胞增殖的影响。结果：HN6细胞对AND-SLNs的药物摄取量显著高于AND。经AND-SLNs和AND分别作用48 h后，HN6细胞的半抑制浓度（IC50）分别为6.09 μg/mL和13.75 μg/mL，Leuk1细胞对2种药物的IC50分别为 0.72 μg/mL和26.14 μg/mL，说明经纳米化后的AND对肿瘤细胞增殖的抑制性明显增强。AND-SLNs和AND作用12 h后，AND-SLNs阻滞周期的活性强于AND，将细胞明显阻滞于G0/G1期（P<0.05）；作用 6 h或12 h后，AND-SLNs诱导细胞凋亡的活性强于AND（P<0.05）。结论：AND-SLNs能促进细胞摄取AND，进而提高AND抑制细胞增殖、阻滞细胞周期、诱导细胞凋亡的活性，具有更强的抗口腔鳞癌和白斑细胞增殖的作用，因此AND-SLNs有望成为防治白斑癌变的新剂型。

关键词: 穿心莲内酯, 穿心莲内酯纳米粒, 口腔白斑, 口腔鳞癌

Abstract:

Objective: To compare inhibitory activity of andrographolide solid lipid nanoparticles(AND-SLNs) and free andrographolide(AND) to cell lines of oral squamous cell carcinoma (HN6 cell line) and oral leukoplakia(Leuk1 cell line). Methods: The drug quantitative uptaken and functional impact about drugs action of HN6 and Leuk1, were examined with high performance liquid chromatography（HPLC）, MTS assay and flow cytometry. Results: Absorption of AND-SLNs in HN6 cells were more than that of AND. The inhibitory activity of AND-SLNs on cell proliferation to HN6 and Leuk1, were significantly more than that of AND. The drugs’ half maximal inhibitory concentration (IC50) after treatment for 48 h were as followed: HN6 48 h IC50 of AND-SLNs was 6.09 μg/mL, while that of AND was 13.75 μg/mL. Leuk1 48 h IC50 of AND-SLNs was 0.72 μg/mL, while that of AND was 26.14 μg/mL. According to 48 h IC50, the bioactivity of AND-SLNs on arresting on cell proliferation cycle at cell phase (G0/G1) was obviously stronger than that of AND（P<0.05）. The biological effects of AND-SLNs on inducing cell apoptosis, after treated by 6 h or 12 h, were obvious stronger than AND（P<0.05）. Conclusion: The bioactivity about inhibition cell proliferation, blocking cell cycle and inducing cell apoptosis of AND-SLNs, were stronger than AND, and AND-SLNs might be a novel drug for prevention and treatment of leukoplakia cancerization.

Key words: andrographolide(AND), andrographolide solid lipid nanoparticles(AND-SLNs), oral leukoplakia, oral squamous cell carcinoma (OSCC)

中图分类号:

李宏权, 姜继宗, 马亭云, 吴玉波, 钱文昊, 王长虹. 穿心莲内酯纳米粒对口腔鳞癌HN6细胞株和白斑Leuk1细胞株抑制作用的实验研究[J]. 《口腔颌面外科杂志》, 2020, 30(4): 216-221.

LI Hongquan, JIANG Jizong, MA Tingyun, WU Yubo, QIAN Wenhao, WANG Changhong. Anticanceration Activity of Andrographolide Solid Lipid Nanoparticles to Oral Squamous Cell Carcinoma HN6 Cell Line and Oral Leukoplakia Leuk1 Cell Line[J]. 《Journal of Oral and Maxillofacial Surgery》, 2020, 30(4): 216-221.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2020/V30/I4/216

图/表 4

图1 HPLC定量检测细胞摄取药物实验注：A. 以不同质量浓度（8、80 μg/mL）的AND-SLNs和AND处理 2 h 后HN6细胞内的AND质量浓度；B. 以不同质量浓度（8、80 μg/mL）的AND-SLNs和AND处理 2 h 后Leuk1细胞内的AND质量浓度。

Figure 1 Cellular uptake in quantitive examination by HPLC

图2 用MTS方法进行细胞增殖抑制实验注：A. 不同浓度AND-SLNs和AND按时间梯度（24、48、72、96 h）处理后，HN6在48 h时的IC50；B. 不同浓度AND-SLNs和AND按时间梯度（24、48、72、96 h）处理后，Leuk1 在48 h的IC50。

Figure 2 Cell viability assay by MTS

图3 流式细胞仪检测细胞周期阻滞实验注：A. 用HN6 48 h IC50 AND-SLNs 6.09 μg/mL处理12 h后HN6的细胞周期阻滞情况；B. 用 HN6 48 h IC50 AND 13.75 μg/mL处理12 h后HN6的细胞周期阻滞情况；C. 用Leuk1 48 h IC50 AND-SLNs 0.72 μg/mL处理12 h后Leuk1的细胞周期阻滞情况；D. 用Leuk1 48 h IC50 AND 26.14 μg/mL处理12 h后Leuk1的细胞周期阻滞情况。

Figure 3 Cell cycle analysis by flow cytometry

图4 Annexin V-PI 凋亡实验注：A、B. HN6经AND-SLNs、AND 48 h IC50处理6 h后；C、D. HN6经AND-SLNs、AND 48 h IC50处理12 h后；E、F. Leuk1经AND-SLNs、AND 48 h IC50处理6 h后；G、H. Leuk1经AND-SLNs、AND 48 h IC50处理12 h后。

Figure 4 Annexin V-PI apoptosis assay

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