藁本内酯调节RhoA/ROCK信号通路对食管癌细胞生物学行为的影响

doi:10.11958/20240758

天津医药 ›› 2024, Vol. 52 ›› Issue (11): 1164-1170.doi: 10.11958/20240758

藁本内酯调节RhoA/ROCK信号通路对食管癌细胞生物学行为的影响

郝凯凯¹(), 王晓敏²^,^△(), 刘峥¹, 刘东洋³, 李静¹

1 河北省邯郸市中心医院肿瘤科（邮编056000）
2 河北工程大学附属医院影像科
3 河北省邯郸市中心医院胸外科

收稿日期:2024-06-11 修回日期:2024-07-19 出版日期:2024-11-15 发布日期:2024-11-12
通讯作者: △E-mail:r61ejj@163.com
作者简介:郝凯凯（1988），男，主治医师，主要从事肿瘤内科学方面研究。E-mail:f93vpv@163.com
基金资助:
河北省2022年度医学科学研究计划(20220505)

Effects of ligustilide regulating RhoA/ROCK signaling pathway on biological behavior of esophageal cancer cells

HAO Kaikai¹(), WANG Xiaomin²^,^△(), LIU Zheng¹, LIU Dongyang³, LI Jing¹

1 Department of Oncology, Handan Central Hospital, Hebei Province, Handan 056000, China
2 Department of Imaging, Affiliated Hospital of Hebei Engineering University
3 Department of Thoracic Surgery, Handan Central Hospital, Hebei Province

Received:2024-06-11 Revised:2024-07-19 Published:2024-11-15 Online:2024-11-12
Contact: △E-mail:r61ejj@163.com

郝凯凯, 王晓敏, 刘峥, 刘东洋, 李静. 藁本内酯调节RhoA/ROCK信号通路对食管癌细胞生物学行为的影响[J]. 天津医药, 2024, 52(11): 1164-1170.

HAO Kaikai, WANG Xiaomin, LIU Zheng, LIU Dongyang, LI Jing. Effects of ligustilide regulating RhoA/ROCK signaling pathway on biological behavior of esophageal cancer cells[J]. Tianjin Medical Journal, 2024, 52(11): 1164-1170.

摘要/Abstract

摘要：

目的探讨藁本内酯（LIG）对食管癌细胞增殖、凋亡、血管生成拟态及Ras同源基因家族蛋白A（Rho A）/Rho关联含卷曲螺旋结合蛋白激酶（ROCK）信号通路的影响。方法用浓度为0、12.5、25、50、100、200 μmol/L LIG处理食管癌细胞EC-109，检测细胞活性，筛选适宜浓度进行后续实验。将EC-109细胞分为对照组（Control组），LIG低、中、高浓度组（LIG-L、LIG-M、LIG-H组），LIG高浓度+RhoA激活剂Naciclasine组（LIG-H+Naciclasine组）。Edu检测细胞增殖，流式细胞术检测细胞凋亡；观察血管生成拟态；Western blot检测细胞增殖、凋亡相关蛋白及RhoA、ROCK蛋白表达，裸鼠移植瘤实验验证LIG对食管癌肿瘤生长的影响，免疫组化检测移植瘤血管内皮生长因子（VEGF）、RhoA、ROCK表达水平。结果与Control组相比，LIG-L、LIG-M、LIG-H组EC-109细胞血管拟态管状结构依次减少，Edu阳性率、细胞周期蛋白（Cyclin） D1、细胞增殖核抗原（Ki67）、B细胞淋巴瘤/白血病-2（Bcl-2）、RhoA、ROCK表达依次降低，P21、细胞凋亡率、Bcl-2相关蛋白（Bax）、胱天蛋白酶（Caspase）-3表达依次升高（P<0.05）。RhoA激活剂Naciclasine可部分逆转LIG对食管癌细胞增殖、凋亡和血管生成拟态的改善作用。裸鼠移植瘤实验显示，与Control组相比，LIG组裸鼠移植瘤生长减缓，肿瘤体积减小，RhoA、ROCK、VEGF表达水平降低（P<0.05）。结论 LIG通过抑制RhoA/ROCK信号通路抑制食管癌细胞的增殖及血管生成拟态，促进食管癌细胞凋亡。

关键词: 藁本内酯, 食管肿瘤, ρA GTP结合蛋白质, rho相关激酶类, 细胞增殖, 细胞凋亡, 肿瘤移植

Abstract:

Objective To investigate effects of ligustilide (LIG) on proliferation, apoptosis, angiogenic mimicry and Ras homolog gene family member A (RhoA)/Rho associated coiled coil containing protein kinase 1 (ROCK) signaling pathway in esophageal cancer cells. Methods Esophageal cancer cell line EC-109 was treated with LIG at concentrations of 0, 12.5, 25, 50, 100, and 200 μmol/L to detect cell activity, and the suitable concentration was selected for subsequent experiments. EC-109 cells were grouped into the control group, the LIG low, medium and high concentration groups (LIG-L, LIG-M and LIG-H groups), and the LIG-H+RhoA activator Naciclassine group (LIG-H+Naciclassine group). Edu was applied to detect cell proliferation, and flow cytometry was applied to detect cell apoptosis. Angiogenetic mimicry was observed. Western blot assay was applied to detect expression levels of proteins related to cell proliferation and apoptosis, and RhoA, ROCK proteins. Nude mouse tumor transplantation experiment was applied to verify the effect of LIG on the growth of esophageal cancer tumors. Immunohistochemistry was applied to detect expression levels of angiogenesis related factors (VEGF), RhoA and ROCK proteins in transplanted tumors. Results Compared with the control group, the vascular mimicry tubular structure of EC-109 cells decreased sequentially in the LIG-L group, the LIG-M group and the LIG-H group. The positive rate of Edu, the expression levels of Cyclin D1, Ki67, Bcl-2, RhoA and ROCK reduced in turn. P21, cell apoptosis rate, the expression of Bax and Caspase-3 increased in sequence (P<0.05). Naciclasine, RhoA activator, partially reversed the effect of LIG on cell proliferation, apoptosis and vasculogenic mimicry of esophageal cancer cells. Nude mouse transplantation tumor experiment showed that compared with the control group, the growth rate of transplanted tumor showed down, tumor volume decreased and the expression levels of RhoA, ROCK and VEGF decreased in the LIG group (P<0.05). Conclusion Ligustilide inhibits the proliferation and angiogenic mimicry of esophageal cancer cells by inhibiting RhoA/ROCK signaling pathway, and promotes the apoptosis of esophageal cancer cells.

Key words: ligustilide, esophageal neoplasms, rhoA GTP-binding protein, rho-associated kinases, cell proliferation, apoptosis, neoplasm transplantation

中图分类号:

R735.1

图/表 13

图1 LIG对EC-109细胞存活率的影响 n=6，F=470.147，P<0.01；a与Control组比较，P<0.05。

Fig.1 Effect of ligustilide on survival rate of EC-109 cells

图2 Edu检测各组EC-109细胞增殖情况（×200）

Fig.2 Proliferation of EC-109 cells in each group tested by Edu (×200)

图3 Western blot检测Cyclin D1、P21、Ki67蛋白表达 A：Control组；B：LIG-L组；C：LIG-M组；D：LIG-H组；E：LIG-H+Naciclasine组

Fig.3 Western blot analysis of Cyclin D1, P21 and Ki67 protein expression

表1 LIG对EC-109细胞增殖及增殖相关蛋白表达的影响（n=6，$\bar{x} \pm s$）

Tab.1 Effects of ligustilide on proliferation and proliferation-related protein expression in EC-109 cells

组别	Edu阳性率/%	Cyclin D1
Control组	42.36±2.14	1.03±0.08
LIG-L组	36.79±1.85^a	0.71±0.07^a
LIG-M组	28.52±1.43^ab	0.48±0.06^ab
LIG-H组	19.48±0.97^abc	0.24±0.04^abc
LIG-H+Naciclasine组	26.13±1.32^d	0.45±0.06^d
F	191.213^＊＊	135.030^＊＊
组别	P21	Ki67
Control组	0.25±0.03	1.21±0.09
LIG-L组	0.37±0.04^a	0.89±0.08^a
LIG-M组	0.69±0.05^ab	0.47±0.07^ab
LIG-H组	0.95±0.07^abc	0.31±0.05^abc
LIG-H+Naciclasine组	0.72±0.08^d	0.48±0.06^d
F	147.387^＊＊	160.494^＊＊

图4 流式细胞术检测各组EC-109细胞凋亡情况

Fig.4 The apoptosis of EC-109 cells in each group detected by flow cytometry

图5 Western blot检测Bax、Bcl-2、Caspase-3蛋白表达 A：Control组；B：LIG-L组；C：LIG-M组；D：LIG-H组；E：LIG-H+Naciclasine组

Fig.5 Western blot analysis of Bax, Bcl-2 and Caspase-3 protein expression

表2 LIG对EC-109细胞凋亡及凋亡相关蛋白表达的影响（n=6，$\bar{x} \pm s$）

Tab.2 Effects of ligustilide on apoptosis and apoptosis-related protein expression in EC-109 cells

组别	细胞凋亡率/%	Bax	Bcl-2	Caspase-3
Control组	3.16±0.25	0.26±0.04	0.76±0.08	0.34±0.04
LIG-L组	20.73±1.82^a	0.48±0.05^a	0.54±0.06^a	0.64±0.05^a
LIG-M组	27.15±2.53^ab	0.80±0.06^ab	0.32±0.05^ab	0.97±0.07^ab
LIG-H组	36.74±3.24^abc	1.15±0.08^abc	0.18±0.04^abc	1.22±0.11^abc
LIG-H+Naciclasine组	25.38±2.36^d	0.83±0.06^d	0.39±0.05^d	0.99±0.06^d
F	176.966^＊＊	199.712^＊＊	88.952^＊＊	143.769^＊＊

图6 各组EC-109细胞血管生成拟态情况（×100）

Fig. 6 Mimicry of angiogenesis of EC-109 cells in each group (×100)

表3 LIG对EC-109细胞血管生成管状结构数及RhoA、ROCK蛋白表达的影响（n=6，$\bar{x} \pm s$）

Tab.3 Effects of ligustilide on angiogenic tubular structure number and protein expression of RhoA and ROCK in EC-109 cells

组别	管状结构数	RhoA	ROCK
Control组	34.50±3.27	1.03±0.09	1.14±0.12
LIG-L组	27.16±2.54^a	0.80±0.08^a	0.91±0.07^a
LIG-M组	20.33±1.83^ab	0.47±0.07^ab	0.58±0.06^ab
LIG-H组	11.83±0.96^abc	0.32±0.05^abc	0.23±0.04^abc
LIG-H+Naciclasine组	18.17±1.62^d	0.54±0.06^d	0.55±0.06^d
F	94.523^＊＊	93.729^＊＊	131.819^＊＊

图7 Western blot检测RhoA、ROCK蛋白表达 A：Control组；B：LIG-L组；C：LIG-M组；D：LIG-H组；E：LIG-H+Naciclasine组。

Fig.7 Western blot analysis of RhoA and ROCK protein expression

表4 裸鼠移植瘤不同时间段体积比较（n=10，cm3，$\bar{x} \pm s$）

Tab.4 Volume comparison of transplanted tumor in nude mice between different time periods

组别	0 d	2 d	4 d	6 d
Control组	0.00±0.00	0.06±0.01	0.20±0.02	0.40±0.04
LIG组	0.00±0.00	0.03±0.01	0.06±0.01	0.12±0.02
t	0.000	6.708^＊＊	19.799^＊＊	19.799^＊＊
组别	8 d	10 d	12 d	14 d
Control组	0.65±0.06	0.87±0.08	1.06±0.10	1.22±0.12
LIG组	0.20±0.02	0.32±0.03	0.44±0.04	0.53±0.05
t	22.500^＊＊	20.356^＊＊	18.204^＊＊	16.784^＊＊

表5 LIG对14 d时裸鼠移植瘤质量及RhoA、ROCK、VEGF蛋白表达的影响（n=10，$\bar{x} \pm s$）

Tab.5 Effects of ligustilide on tumor quality and protein expression of RhoA, ROCK and VEGF in nude mice at 14 days

组别	移植瘤质量/g	RhoA/OD值	ROCK/OD值	VEGF/OD值
Control组	1.37±0.38	0.91±0.09	0.82±0.08	0.76±0.10
LIG组	0.59±0.17	0.46±0.05	0.38±0.04	0.25±0.06
t	5.925^＊＊	13.822^＊＊	15.556^＊＊	13.829^＊＊

图8 免疫组化检测移植瘤组织中RhoA、ROCK、VEGF蛋白表达（×400）

Fig.8 Immunohistochemical detection of RhoA, ROCK and VEGF protein expression in transplanted tumor tissue (×400)

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藁本内酯调节RhoA/ROCK信号通路对食管癌细胞生物学行为的影响

Effects of ligustilide regulating RhoA/ROCK signaling pathway on biological behavior of esophageal cancer cells

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