紫草素调节Notch信号通路对肝癌细胞恶性生物学活性的影响

doi:10.11958/20221929

天津医药 ›› 2023, Vol. 51 ›› Issue (12): 1293-1299.doi: 10.11958/20221929

紫草素调节Notch信号通路对肝癌细胞恶性生物学活性的影响

吴德建¹(), 杨秋², 谢桂丹¹, 彭鑫¹^,^△()

1.儋州市人民医院消化内科（邮编571799）
2.儋州市人民医院检验科（邮编571799）

收稿日期:2022-11-22 修回日期:2023-03-20 出版日期:2023-12-15 发布日期:2023-12-22
通讯作者: ^△ E-mail：gjocp60@163.com
作者简介:吴德建（1993），男，主治医师，主要从事肝炎、肝硬化诊疗方面研究。E-mail：wudejian196306@163.com

Impact of shikonin on the malignant biological activity of liver cancer cells by regulating Notch signaling pathway

WU Dejian¹(), YANG Qiu², XIE Guidan¹, PENG Xin¹^,^△()

1. Department of Gastroenterology, Danzhou People's Hospital, Danzhou 571799, China
2. Department of Laboratory, Danzhou People's Hospital, Danzhou 571799, China

Received:2022-11-22 Revised:2023-03-20 Published:2023-12-15 Online:2023-12-22
Contact: ^△ E-mail：gjocp60@163.com

吴德建, 杨秋, 谢桂丹, 彭鑫. 紫草素调节Notch信号通路对肝癌细胞恶性生物学活性的影响[J]. 天津医药, 2023, 51(12): 1293-1299.

WU Dejian, YANG Qiu, XIE Guidan, PENG Xin. Impact of shikonin on the malignant biological activity of liver cancer cells by regulating Notch signaling pathway[J]. Tianjin Medical Journal, 2023, 51(12): 1293-1299.

摘要/Abstract

摘要：

目的探讨紫草素（SHI）调节Notch信号通路对肝癌细胞恶性生物学活性的影响。方法采用Western blot分别检测肝癌组织、癌旁组织、肝癌细胞（HepG2、Hep3B、HCCLM3、Huh-7、SMMC-7721细胞）及正常肝细胞（HL-7702细胞）中Notch、发状分裂相关增强子-1（HES1）、hairy相关转录因子1（HEY1）蛋白表达；将Huh-7细胞分为对照组、L-SHI组（1 μmol/L SHI）、M-SHI组（2 μmol/L SHI）、H-SHI组（4 μmol/L SHI）、DAPT组（50 μmol/L Notch信号抑制剂DAPT）、H-SHI+丙戊酸（VPA）组（4 μmol/L SHI和8 mmol/L Notch通路激活剂VPA）。CCK-8法和平板克隆实验检测Huh-7细胞增殖；流式细胞术检测Huh-7细胞凋亡以及细胞周期；细胞划痕实验以及Transwell侵袭实验分别检测Huh-7细胞迁移和侵袭情况；Western blot检测上皮间质转化（EMT）及凋亡相关蛋白表达。结果 Notch、HES1、HEY1在肝癌组织和细胞中表达水平明显升高，且Huh-7细胞差异最明显，以Huh-7细胞为研究对象。与对照组相比，L-SHI组、M-SHI组、H-SHI组、DAPT组Notch、HES1、HEY1、Bcl-2蛋白表达水平下调，S期和G2/M期细胞比例、OD₄₅₀值及克隆数、迁移率、侵袭细胞数目、N-cadherin、Vimentin水平降低（P<0.05），G1/G0期细胞比例、凋亡率、Bax、cleaved-Caspase-3、E-cadherin水平升高（P<0.05），且SHI的作用效果呈现剂量依赖性；与H-SHI组相比，H-SHI+VPA组以上指标趋势相反，VPA减弱了SHI降低肝癌细胞恶性生物学活性的作用。结论 SHI可能通过抑制Notch信号通路抑制Huh-7细胞增殖、迁移和侵袭，促进Huh-7细胞凋亡。

关键词: 紫草素, 癌, 肝细胞, 受体, Notch, 细胞增殖, 细胞运动, 细胞凋亡

Abstract:

Objective To investigate the impact of shikonin (SHI) on the malignant biological activity of liver cancer cells by regulating Notch signaling pathway. Methods Western blot assay was used to detect the expression of Notch, Hairy mitosis-related enhancer-1 (Hes1), hairy-related transcription factor-1 (HEY1) protein in liver cancer tissue, paracancerous tissue, hepatoma cells (HepG2 cells, Hep3B cells, HCCLM3 cells, Huh-7 cells and SMMC-7721 cells) and normal liver cells (HL-7702 cells). Huh-7 cells were divided into the control group, the L-SHI group (1 μmol/L SHI), the M-SHI group (2 μmol/L SHI), the H-SHI group (4 μmol/L SHI), the DAPT group (50 μmol/L Notch signal inhibitor DAPT) and the H-SHI+VPA group [4 μmol/L SHI and 8 mmol/L Notch pathway activator Valproic acid (VPA)]. The proliferation of Huh-7 cells was detected by CCK-8 method and plate cloning test. The apoptosis and cell cycle of Huh-7 cells were detected by flow cytometry. Cell scratch test and Transwell invasion test were used to detect migration and invasion of Huh-7 cells. Western blot assay was used to detect the expression of epithelial-mesenchymal transformation (EMT) and apoptosis related proteins. Results The expression levels of Notch, HES1 and HEY1 were obviously increased in liver cancer tissue and cells, and Huh-7 cells showed the most obvious difference, therefore, Huh-7 cells were taken as the research object. Compared with the control group, the protein levels of Notch, HES1, HEY1 and Bcl-2 decreased, and the proportions of S phase and G2 phase cells, OD₄₅₀ value, number of clones, migration rate, number of invasive cells and levels of N-cadherin and Vimentin decreased significantly in the L-SHI group, the M-SHI group, the H-SHI group and the DAPT group (P<0.05). The proportion of G1/G0 phase cells, apoptosis rate and levels of Bax, cleaved Casase-3, and E-cadherin increased obviously (P<0.05). The effect of SHI was dose-dependent. Compared with the H-SHI group, the above indexes showed the opposite trend in the H-SHI+VPA group. VPA attenuated the effect of SHI on reducing the malignant biological activity of liver cancer cells. Conclusion SHI may inhibit the proliferation, migration and invasion of Huh-7 cells and promote apoptosis of Huh-7 cells by inhibiting Notch signal pathway.

Key words: Shikonin, carcinoma, hepatocellular, receptors, Notch, cell proliferation, cell movement, apoptosis

中图分类号:

R735.7

图/表 18

表1 Notch、HES1、HEY1蛋白在癌组织及癌旁组织中的表达

Tab.1 Expression of Notch, HES1 and HEY1 proteins in liver cancer tissue and adjacent tissue （n=20，$\bar{x}±s$）

组别	Notch	HES1	HEY1
癌旁组织	0.78±0.09	0.63±0.07	0.39±0.05
癌组织	1.41±0.13	1.24±0.11	1.37±0.14
t	17.819^＊＊	10.633^＊＊	29.481^＊＊

表2 Notch、HES1、HEY1蛋白在正常肝细胞及肝癌细胞中的表达

Tab.2 Expression of Notch, HES1 and HEY1 proteins in normal liver cells and liver cancer cells （n=6，$\bar{x}±s$）

组别	Notch	HES1	HEY1
HL-7702细胞	0.60±0.06	0.51±0.05	0.35±0.04
HepG2细胞	1.41±0.13^a	1.05±0.09^a	1.16±0.12^a
Hep3B细胞	1.33±0.12^a	1.13±0.12^a	1.22±0.13^a
HCCLM3细胞	1.35±0.14^a	1.07±0.11^a	1.26±0.14^a
SMMC-7721细胞	1.37±0.15^a	1.09±0.13^a	1.24±0.16^a
Huh-7细胞	1.60±0.17^a	1.23±0.14^a	1.42±0.17^a
F	67.663^＊＊	52.880^＊＊	81.609^＊＊

图1 Western blot检测肝癌组织及癌旁组织中Notch、HES1、HEY1蛋白表达

Fig.1 Western blot detection of Notch, HES1 and HEY1 protein expression in liver cancer tissue and adjacent tissue

图2 Western blot检测正常肝细胞及肝癌细胞中Notch、HES1、HEY1蛋白水平 A：HL-7702细胞；B：HepG2细胞；C：Hep3B细胞；D：HCCLM3细胞；E：SMMC-7721细胞；F：Huh-7细胞。

Fig.2 Western blot detection of Notch, HES1 and HEY1 protein levels in normal hepatocytes and hepatoma cells

图3 Western blot检测SHI对Huh-7细胞中Notch、HES1、HEY1蛋白的影响 A：对照组；B：L-SHI组；C：M-SHI组；D：H-SHI组；E：DAPT组；F：H-SHI+VPA组。

Fig.3 Western blot detection of effects of SHI on Notch, HES1 and HEY1 proteins in Huh-7 cells

表3 SHI对Huh-7细胞中Notch、HES1、HEY1蛋白表达的影响

Tab.3 Effects of SHI on Notch, HES1 and HEY1 proteins in Huh-7 cells （n=6，$\bar{x}±s$）

组别	Notch	HES1	HEY1
对照组	1.63±0.19	1.26±0.14	1.48±0.17
L-SHI组	1.31±0.15^a	1.03±0.12^a	1.15±0.13^a
M-SHI组	0.98±0.10^ab	0.80±0.09^ab	0.79±0.09^ab
H-SHI组	0.64±0.06^abc	0.63±0.08^abc	0.38±0.04^abc
DAPT组	0.72±0.07^a	0.39±0.03^a	0.89±0.09^a
H-SHI+VPA组	1.39±0.14^d	1.11±0.14^d	1.27±0.15^d
F	96.630^＊＊	91.860^＊＊	106.778^＊＊

图4 SHI对Huh-7细胞克隆形成的影响

Fig.4 Effects of SHI on the formation of Huh-7 cell clone

表4 SHI对Huh-7细胞OD450值及克隆数的影响

Tab.4 Effects of SHI on OD450 value and clone number of Huh-7 cells

组别	OD₄₅₀值	克隆数/个
对照组	1.33±0.14	56.76±5.94
L-SHI组	1.12±0.11^a	44.83±5.54^a
M-SHI组	0.89±0.09^ab	35.37±4.65^ab
H-SHI组	0.67±0.07^abc	24.16±2.21^abc
DAPT组	0.70±0.06^a	26.73±2.02^a
H-SHI+VPA组	1.18±0.12^d	45.17±5.47^d
F	69.795^＊＊	32.725^＊＊

图5 流式细胞术检测SHI对Huh-7细胞细胞周期的影响

Fig.5 Effects of SHI on cell cycle of Huh-7 cells by flow cytometry

图6 流式细胞术检测Huh-7细胞凋亡

Fig.6 Detection of Huh-7 cell apoptosis by flow cytometry

图7 SHI对Huh-7细胞中Bcl-2、Bax、cleaved-Caspase-3蛋白水平的影响 A：对照组；B：L-SHI组；C：M-SHI组；D：H-SHI组；E：DAPT组；F：H-SHI+VPA组。

Fig.7 Effects of SHI on Bcl-2, Bax, cleaved-Caspase-3 protein levels in Huh-7 cells

表5 SHI对Huh-7细胞细胞周期的影响

Tab.5 Effects of SHI on cell cycle of Huh-7 cells

组别	细胞周期分布
组别	G1/G0	S	G2/M
对照组	44.13±4.27	38.45±3.56	17.42±1.69
L-SHI组	51.01±5.53^a	33.65±3.16^a	15.34±1.79^a
M-SHI组	52.89±5.76^a	31.92±3.03^a	15.19±1.57^a
H-SHI组	58.34±5.65^a	26.53±2.45^a	15.13±1.35^a
DAPT组	57.23±5.34^a	27.96±2.67^a	14.81±1.23^a
H-SHI+VPA组	50.78±5.56^d	33.12±3.32^d	16.10±1.37^d
F	9.124^＊＊	20.200^＊＊	7.609^＊＊

表6 SHI对Huh-7细胞凋亡率和凋亡蛋白的影响

Tab.6 Effects of SHI on apoptosis rate and apoptotic protein of Huh-7 cells

组别	凋亡率/%	Bcl-2	Bax	cleaved- Caspase-3
对照组	2.43±0.27	1.45±0.17	0.32±0.02	0.33±0.03
L-SHI组	5.35±0.52^a	1.11±0.12^a	0.73±0.06^a	0.69±0.07^a
M-SHI组	8.65±0.98^ab	0.82±0.09^ab	1.04±0.11^ab	0.99±0.09^ab
H-SHI组	11.65±1.23^abc	0.53±0.05^abc	1.36±0.14^abc	1.37±0.14^abc
DAPT组	9.97±0.96^a	0.46±0.04^a	1.30±0.13^a	1.28±0.12^a
H-SHI+ VPA组	4.78±0.46^d	1.17±0.12^d	0.67±0.07^d	0.57±0.06^d
F	187.108^＊＊	129.293^＊＊	168.904^＊＊	197.238^＊＊

图8 SHI对Huh-7细胞侵袭细胞数量的影响（×200）

Fig.8 Effects of SHI on the number of invasive cells of Huh-7 cells（×200）

图9 SHI对Huh-7细胞迁移率的影响

Fig.9 Effects of SHI on the migration rate of Huh-7 cells

图10 Western blot检测Huh-7细胞中E-cadherin、N-cadherin、Vimentin蛋白水平 A：对照组；B：L-SHI组；C：M-SHI组；D：H-SHI组；E：DAPT组；F：H-SHI+VPA组。

Fig.10 Western blot detection of E-cadherin, N-cadherin and Vimentin protein levels in Huh-7 cells

表7 SHI对Huh-7细胞迁移率和侵袭细胞数目的影响

Tab.7 Effects of SHI on migration rate and number of invasive cells of Huh-7 cells

组别	迁移率/%	侵袭细胞数目/（个/视野）
对照组	30.22±3.32	205.43±20.25
L-SHI组	23.94±2.37^a	183.35±19.48^a
M-SHI组	16.74±1.70^ab	166.33±16.46^ab
H-SHI组	10.86±1.23^abc	141.57±15.13^abc
DAPT组	12.13±1.26^a	150.73±15.97^a
H-SHI+VPA组	25.48±2.76^d	199.87±20.42^d
F	121.414^＊＊	20.769^＊＊

表8 SHI对Huh-7细胞E-cadherin、N-cadherin、Vimentin表达水平的影响

Tab.8 Effects of SHI on levels of E-cadherin, N-cadherin and Vimentin in Huh-7 cells

组别	E-cadherin	N-cadherin	Vimentin
对照组	0.22±0.02	0.90±0.09	1.18±0.21
L-SHI组	0.39±0.04^a	0.71±0.07^a	0.98±0.09^a
M-SHI组	0.57±0.06^ab	0.52±0.05^ab	0.74±0.08^ab
H-SHI组	0.78±0.09^abc	0.30±0.02^abc	0.52±0.05^abc
DAPT组	0.72±0.07^a	0.35±0.03^a	0.58±0.06^a
H-SHI+VPA组	0.36±0.03^d	0.79±0.09^d	1.04±0.13^d
F	148.635^＊＊	143.108^＊＊	52.235^＊＊

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紫草素调节Notch信号通路对肝癌细胞恶性生物学活性的影响

Impact of shikonin on the malignant biological activity of liver cancer cells by regulating Notch signaling pathway

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