预知子提取物增敏奥沙利铂诱导结肠癌HCT116细胞凋亡的研究

周婷婷; 沈卫星; 陈桐庆; 冯慧; 谭佳妮; 徐长亮; 余成涛; 范旻旻; 程海波

doi:10.14148/j.issn.1672-0482.2022.0204

预知子提取物增敏奥沙利铂诱导结肠癌HCT116细胞凋亡的研究

doi: 10.14148/j.issn.1672-0482.2022.0204

周婷婷^1,,
沈卫星^{1, 2, ,},
陈桐庆¹,
冯慧¹,
谭佳妮^{1, 2},
徐长亮^{1, 2},
余成涛^{1, 2},
范旻旻^{1, 2},
程海波^{1, 2}

1.
南京中医药大学第一临床医学院, 江苏南京 210023
2.
江苏省中医药防治肿瘤协同创新中心, 江苏南京 210023

基金项目:

国家自然科学基金面上项目 81973523

详细信息

作者简介:
周婷婷, 女, 硕士研究生, E-mail: 20200056@njucm.edu.cn

通讯作者:
沈卫星, 男, 副教授, 主要从事中医药防治肿瘤的临床研究, E-mail: weixingshen@njucm.edu.cn

中图分类号: R285.5
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- 被引次数: 0
出版历程
- 收稿日期: 2021-09-09
- 网络出版日期: 2022-03-24
- 发布日期: 2022-03-10

Study on Fructus Akebiae Extraction Inducing Apoptosis Sensitization of Colon Cancer HCT116 Cells to Oxaliplatin

ZHOU Ting-ting^1
,,
SHEN Wei-xing^{1, 2
, ,},
CHEN Tong-qing¹,
FENG Hui¹,
TAN Jia-ni^{1, 2},
XU Chang-liang^{1, 2},
YU Cheng-tao^{1, 2},
FAN Min-min^{1, 2},
CHENG Hai-bo^{1, 2}

1.
The First College of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine in Prevention and Treatment of Tumor, Nanjing University of Chinese Medicine, Nanjing 210023, China

摘要

摘要: 目的探讨预知子提取物增敏奥沙利铂(OXA)诱导结肠癌HCT116细胞凋亡的作用及机制, 为减少OXA毒副作用发生的临床应用提供研究基础。方法将预知子乙酸乙酯萃取成分进行中压柱层析分离, 并进行抗肿瘤药效筛选, 获取活性部位B14, 并进一步分离和鉴定该活性部位成分; 运用MTT法检测B14、OXA及二者联用对HCT116的增殖抑制作用；采用CompuSyn软件评价联合药效, 确定药物联用最佳剂量; 流式细胞术检测B14与OXA联用后HCT116细胞的凋亡率及周期分布; Western blot法检测凋亡及信号通路相关蛋白的表达水平。结果筛选确定预知子活性部分B14，检测并明确其主要成分为皂苷B和α-常春藤皂苷；6 μg·mL^-1 B14可显著促进15 μg·mL^-1 OXA对HCT116细胞的增殖抑制作用(P < 0.001)，促进细胞凋亡(P < 0.001)；联合组细胞周期被阻滞于G2/M期(P < 0.001)；Western blot结果显示，与OXA组比较，联合组Bcl-2、Caspase-3及Bcl-2/Bax比值显著降低(P < 0.01，P < 0.001)，Bax、Cyt-c、Cleaved caspase-3、Cleaved caspase-3/Caspase-3比值及p-p38 MAPK显著升高(P < 0.05，P < 0.01，P < 0.001)。结论预知子提取物具有增敏OXA诱导HCT116凋亡的作用, 其机制可能与阻滞细胞周期在G₂/M期及激活p38 MAPK介导的线粒体凋亡通路有关。研究结果为降低OXA毒副作用的临床应用提供了线索。
- 预知子 /
- 奥沙利铂 /
- 结肠癌 /
- HCT116 /
- 增敏 /
- 凋亡
Abstract: OBJECTIVE To investigate the efficacy and mechanism of Fructus akebiae extraction (B14) inducing the apoptosis sensitization of colon cancer HCT116 cells to oxaliplatin (OXA), and provide the evidence for the prevention of side effect of OXA in clinical application. METHODS The ethyl acetate extraction of Fructus akebiae were isolated by medium pressure column chromatography, and the active fraction B14 was obtained by anti-tumor experiment, further followed by research for its component identification. MTT assay was used to detect the proliferation inhibition of HCT116 by B14, OXA and their combination. CompuSyn software was used to evaluate the sensitization efficiency and determine the optimal dose of the combination. The apoptosis rate and cycle distribution of HCT116 cells were detected by flow cytometry. The expressions of apoptosis-related proteins were detected by Western blot. RESULTS The main components of the active fraction B14 were identified as saponin B and α-hederin. 6 μg·mL^-1 B14 combinated with 15 μg·mL^-1 OXA significantly inhibited the proliferation and promoted the apoptosis of HCT116 cells (P < 0.001). The cell cycle of HCT116 was arrested at G₂/M stage after the combination treatment (P < 0.001). Western blot results showed that, compared with OXA group, protein expressions of Bcl-2, Caspase-3 and the ratio of Bcl-2/Bax in combination group were significantly down-regulated (P < 0.01, P < 0.001), while Bax, Cyt-c, Cleaved caspase-3, the ratio of Cleaved caspase-3/Caspase-3 and p-p38 MAPK were significantly up-regulated (P < 0.05, P < 0.01, P < 0.001). CONCLUSION The extraction of Fructus akebiae can induce the apoptosis sensitization of HCT116 cells to OXA, which may be related to the regulation of p38 MAPK pathway. This study may provide the evidence for preventing the side effect of OXA in clinical application.
- Fructus akebiae /
- oxaliplatin /
- colon cancer /
- HCT116 /
- sensitization /
- apoptosis

HTML全文

图 1 标准品色谱图

Figure 1. The HPLC chromatograms of standard products

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图 2 B14的HPLC图谱

注：1.皂苷B；2.α-常春藤皂苷

Figure 2. The HPLC chromatogram of B14

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图 3 B14对HCT116细胞增殖的作用

Figure 3. The effect of B14 on HCT116 cells proliferation

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图 4 不同浓度B14+OXA抑制HCT116细胞增殖的联合指数图

Figure 4. Fa-CI plot of HCT116 cells proliferation inhibition with different concentrations of B14+OXA

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图 5 各组HCT116细胞形态比较(×100)

A.空白组; B.B14组; C.OXA组; D.联合组

Figure 5. HCT116 cell morphology in each group (×100)

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图 6 各组HCT116细胞凋亡的比较

注：与对照组比较, ^{* *}P < 0.01, ^{* * *}P < 0.001。x±s, n=3。

Figure 6. The apoptosis rates of HCT116 cells in each group

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图 7 各组HCT116细胞周期的比较

注：与对照组比较, ^*P < 0.05, ^{* * *}P < 0.001。x±s, n=3。

Figure 7. The cycle distribution of HCT116 cells in each group

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图 8 各组p38 MAPK信号通路及凋亡相关蛋白的表达情况

注: A.对照组; B.B14组; C.OXA组; D.联合组; 与对照组比较, ^*P < 0.05, ^{* *}P < 0.01, ^{* * *}P < 0.001; 与OXA组比较, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001。x±s, n=3。

Figure 8. The expressions of p38 MAPK signal pathway and apoptosis related proteins in each group

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表 1 各组HCT116细胞增殖抑制率的比较(x±s，%, n=3)

Table 1. Comparison of inhibitory rates of HCT116 cells proliferation in each group (x±s, %, n=3)

B14	OXA
B14	0	7.5 μg·mL^-1	15 μg·mL^-1	25 μg·mL^-1	40 μg·mL^-1	80 μg·mL^-1
0	0.02±1.34	11.75±2.86^***	18.36±3.12^***	27.93±2.42^***	38.74±2.61^***	54.83±3.07^***
3 μg·mL^-1	-1.8±1.57	15.47±2.19^***#	20.96±1.44^***	32.79±1.73^***#	41.36±1.48^***	60.27±2.53^***#
6 μg·mL^-1	-3.53±2.14	23.24±1.35^***###	48.46±2.41^***###	51.88±1.19^***###	62.76±1.81^***###	72.71±1.63^***###
12 μg·mL^-1	20.62±1.44^###	40.29±2.23^***###	55.82±1.94^***###	68.17±2.75^***###	74.13±1.72^***###	83.26±1.58^***###
24 μg·mL^-1	67.73±2.71^###	80.85±2.38^***###	87.74±1.39^***###	92.95±1.93^***###	96.11±2.96^***###	97.68±2.15^***###
32 μg·mL^-1	85.94±1.55^###	95.95±2.79^***###	96.58±1.26^***###	99.87±2.34^***###	98.03±2.19^***###	99.78±1.93^***###
注: 与B14单药组(OXA=0)比较, ^{* * *}P < 0.001;与OXA单药组(B14=0)比较, ^#P < 0.05, ^###P < 0.001。

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