人参皂苷Rg3通过抑制mTOR通路介导的磷酸戊糖途径对肺癌细胞的放射增敏作用

doi:10.11958/20222018

天津医药 ›› 2023, Vol. 51 ›› Issue (8): 791-796.doi: 10.11958/20222018

人参皂苷Rg3通过抑制mTOR通路介导的磷酸戊糖途径对肺癌细胞的放射增敏作用

黄琳(), 李彬, 胡作为^△()

武汉市第一医院肿瘤科（邮编430000）

收稿日期:2022-12-08 修回日期:2023-02-10 出版日期:2023-08-15 发布日期:2023-08-10
通讯作者: ^△E-mail：827823053@qq.com
作者简介:黄琳（1982），女，主治医师，主要从事胸部肿瘤放化疗、靶向治疗方面研究。E-mail：huanglin1982h@163.com
基金资助:
武汉市医学科研项目(WZ20Q04)

The radiosensitizing effect of ginsenoside Rg3 on lung cancer cells by inhibiting mTOR pathway-mediated pentose phosphate pathway

HUANG Lin(), LI Bin, HU Zuowei^△()

Department of Oncology, Wuhan NO.1 Hospital, Wuhan 430000, China

Received:2022-12-08 Revised:2023-02-10 Published:2023-08-15 Online:2023-08-10
Contact: ^△E-mail: 827823053@qq.com

黄琳, 李彬, 胡作为. 人参皂苷Rg3通过抑制mTOR通路介导的磷酸戊糖途径对肺癌细胞的放射增敏作用[J]. 天津医药, 2023, 51(8): 791-796.

HUANG Lin, LI Bin, HU Zuowei. The radiosensitizing effect of ginsenoside Rg3 on lung cancer cells by inhibiting mTOR pathway-mediated pentose phosphate pathway[J]. Tianjin Medical Journal, 2023, 51(8): 791-796.

摘要/Abstract

摘要：

目的探讨人参皂苷Rg3通过抑制哺乳动物雷帕霉素靶蛋白（mTOR）通路介导的磷酸戊糖途径（PPP），对肺癌细胞放射敏感性的影响。方法以0、10、20、40、60、80 mg/L的人参皂苷Rg3处理肺癌细胞A549；MTT法检测细胞增殖情况；将细胞分为对照组（正常培养，不照射）、放射组（X射线照射处理）、人参皂苷Rg3组（60 mg/L人参皂苷Rg3，不照射）、联合组（X射线照射+60 mg/L人参皂苷Rg3）、激活剂组（X射线照射+60 mg/L人参皂苷Rg3+100 nmol/L mTOR通路激活剂MHY1485）；均为加入相对应药物培养48 h后放射组、联合组和激活剂组采用8 Gy X射线照射。平板克隆实验检测各组细胞克隆形成率；酶联免疫吸附试验（ELISA）测定各组细胞葡萄糖-6-磷酸脱氢酶（G6PD）、还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）水平；DCFH-DA荧光探针法检测细胞内活性氧（ROS）水平；流式细胞仪检测细胞凋亡；γ-H2AX免疫荧光染色分析DNA损伤修复情况；Western blot检测细胞中mTOR、p-mTOR、增殖细胞核抗原（PCNA）、Bcl-2相关X蛋白（Bax）、胱天蛋白酶3（caspase-3）、γ-H2AX蛋白的表达。结果人参皂苷Rg3以剂量依赖性的方式抑制A549细胞的增殖（P<0.05）；与对照组比较，放射组、人参皂苷Rg3组细胞克隆形成率、G6PD、ROS、NADPH水平下降，p-mTOR/mTOR、PCNA蛋白表达水平降低，细胞凋亡率、γ-H2AX焦点数、Bax、caspase-3、γ-H2AX蛋白表达水平升高（P<0.05）；与放射组、人参皂苷Rg3组比较，联合组细胞克隆形成率、G6PD、ROS、NADPH水平下降，p-mTOR/mTOR、PCNA蛋白表达水平降低，细胞凋亡率、γ-H2AX焦点数、Bax、caspase-3、γ-H2AX蛋白表达升高（P<0.05）；与联合组比较，激活剂组细胞克隆形成率、G6PD、ROS、NADPH水平升高，p-mTOR/mTOR、PCNA蛋白表达水平升高，细胞凋亡率、γ-H2AX焦点数、Bax、caspase-3、γ-H2AX蛋白表达水平降低（P<0.05）。结论人参皂苷Rg3发挥抑制肺癌作用可能是通过抑制mTOR介导的PPP实现的。

关键词: 人参皂苷Rg3, 肺肿瘤, 辐射耐受性, 辐射增敏药, 磷酸戊糖途径, 哺乳动物雷帕霉素靶蛋白

Abstract:

Objective To investigate the influence of ginsenoside Rg3 on the radiosensitivity of lung cancer cells by inhibiting the pentose phosphate pathway (PPP) mediated by mammalian target of rapamycin (mTOR) pathway. Methods A549 lung cancer cells were treated with 0, 10, 20, 40, 60, 80 mg/L ginsenoside Rg3. The proliferation of A549 cells was detected by MTT method. Cells were divided into the control group (normal culture, no irradiation), the radiation group (X-ray irradiation), the ginsenoside Rg3 group (60 mg/L ginsenoside Rg3, no irradiation), the combination group (X-ray irradiation +60 mg/L ginsenoside Rg3) and the activator group (X-ray irradiation +60 mg/L ginsenoside Rg3+100 nmol/L mTOR pathway activator MHY1485). All of groups were irradiated by 8 GyX ray after 48 h of culture with corresponding drugs. Plate cloning experiment was applied to detect the formation rate of cell clones in each group. Enzyme-linked immunosorbent assay (ELISA) was applied to determine levels of glucose-6-phosphate dehydrogenase (G6PD) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in cell supernatant of each group. DCFH-DA fluorescent probe method was applied to detect the level of intracellular reactive oxygen species (ROS). Cell apoptosis was detected by flow cytometry. γ-H2AX immunofluorescence staining was applied to analyze DNA damage repair. Western blot assay was applied to detect expression levels of mTOR, p-mTOR, proliferating cell nuclear antigen (PCNA), Bcl-2-associated X protein (Bax), caspase-3 and γ-H2AX protein in cells. Results Ginsenoside Rg3 inhibited the proliferation of A549 cells in a dose-dependent manner (P<0.05). Compared with the control group, the cell clone formation rate, G6PD, ROS, NADPH levels, p-mTOR/mTOR and PCNA protein expressions were significantly decreased in the radiation group and the ginsenoside Rg3 group, and the cell apoptosis rate, γ-H2AX foci number, Bax, caspase-3, γ-H2AX protein expressions were significantly increased (P<0.05). Compared with the radiation group and the ginsenoside Rg3 group, the cell clone formation rate, G6PD, ROS, NADPH levels, p-mTOR/mTOR, and PCNA protein expressions were significantly decreased in the combination group, the cell apoptosis rate, γ-H2AX foci number, Bax, caspase-3, γ-H2AX protein expressions were significantly increased (P<0.05). Compared with the combination group, the cell clone formation rate, G6PD, ROS, NADPH levels, p-mTOR/mTOR and PCNA protein expressions were significantly increased in the activator group, and the cell apoptosis rate, γ-H2AX foci number, Bax, caspase-3, γ-H2AX protein expressions were significantly decreased (P<0.05). Conclusion The inhibitory effect of ginsenoside Rg3 on lung cancer cells may be realized through inhibition of PPP mediated by mTOR.

Key words: Ginsenoside Rg3, lung neoplasms, radiation tolerance, radiation-sensitizing agents, pentose phosphate pathway, mammalian target of rapamycin

中图分类号:

R285，R734.2

图/表 5

图1 各组A549细胞凋亡流式图

Fig.1 Flow cytometry of A549 cell apoptosis in each group

表1 各组A549细胞内G6PD、ROS、NADPH水平比较（n=6，$\bar{x}±s$）

Tab.1 Comparison of G6PD, ROS and NADPH levels between the five groups of A549 cells

组别	G6PD/（U/L）	ROS/AU	NADPH/（ng/L）
对照组	3.56±0.42	37.94±2.86	98.64±7.51
放射组	2.87±0.34^a	29.65±2.43^a	76.52±6.64^a
人参皂苷Rg3组	2.31±0.25^a	21.52±2.14^a	63.18±5.62^a
联合组	1.12±0.13^bc	15.61±1.84^bc	33.84±5.16^bc
激活剂组	2.54±0.32^d	26.87±2.23^d	56.87±6.12^d
F	50.664^＊＊	78.867^＊＊	87.996^＊＊

图2 各组A549细胞γ-H2AX焦点形成图像（×1 000）蓝色（DAPI）为细胞核，绿色（γ-H2AX）为DNA双链断裂点。

Fig.2 Image of γ-H2AX foci formation in A549 cells of each group (×1 000)

图3 各组mTOR、p-mTOR、PCNA、Bax、caspase-3、γ-H2AX蛋白表达印迹图 A：对照组；B：放射组；C：人参皂苷Rg3组；D：联合组；E：激活剂组。

Fig.3 Western blot results of mTOR, p-mTOR, PCNA, Bax, caspase-3 and γ-H2AX protein expression in each group

表2 各组A549细胞中PCNA、Bax、caspase-3、γ-H2AX蛋白表达比较（n=6，$\bar{x}±s$）

Tab.2 Comparison of PCNA, Bax, caspase-3 and γ-H2AX expression between the five groups of A549 cells

组别		p-mTOR/mTOR		PCNA
对照组		0.86±0.08		1.68±0.15
放射组		0.53±0.04^a		1.15±0.11^a
人参皂苷Rg3组		0.48±0.04^a		0.87±0.08^a
联合组		0.24±0.03^bc		0.26±0.04^bc
激活剂组		0.61±0.07^d		0.98±0.08^d
F		97.851^＊＊		160.573^＊＊
组别	Bax		caspase-3		γ-H2AX
对照组	0.32±0.04		0.34±0.05		0.28±0.01
放射组	0.71±0.06^a		0.75±0.08^a		0.73±0.07^a
人参皂苷Rg3组	1.08±0.10^a		1.13±0.11^a		0.86±0.09^a
联合组	1.53±0.12^bc		1.62±0.13^bc		1.71±0.14^bc
激活剂组	0.72±0.08^d		0.81±0.09^d		0.75±0.06^d
F	172.975^＊＊		148.533^＊＊		224.736^＊＊

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人参皂苷Rg3通过抑制mTOR通路介导的磷酸戊糖途径对肺癌细胞的放射增敏作用

The radiosensitizing effect of ginsenoside Rg3 on lung cancer cells by inhibiting mTOR pathway-mediated pentose phosphate pathway

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