基于PI3K/Akt/mTOR信号通路探讨黄芪多糖对结直肠癌自噬的影响

doi:10.11958/20220463

天津医药 ›› 2023, Vol. 51 ›› Issue (3): 240-245.doi: 10.11958/20220463

基于PI3K/Akt/mTOR信号通路探讨黄芪多糖对结直肠癌自噬的影响

郅强¹(), 张楠^△(), 冯光玲¹, 孙维义², 赵媛媛¹, 杨世发¹

1 河南中医药大学（邮编450000）
2 河南中医药大学第一附属医院微创腔镜外科

收稿日期:2022-04-01 修回日期:2022-09-02 出版日期:2023-03-15 发布日期:2023-03-02
通讯作者: 张楠 E-mail:zqhnzyydx@163.com;znhnzyydx@163.com
作者简介:郅强（1994），男，硕士在读，主要从事普外科肿瘤相关疾病的临床与实验方面研究。E-mail：zqhnzyydx@163.com
基金资助:
河南省中医管理局科研基金项目(2019ZY2029);河南省卫生健康委重大专项(2022ZYZD02)

Study on the mechanism of astragalus polysaccharides regulating autophagy of colorectal cancer cells based on PI3K/Akt/mTOR signaling pathway

ZHI Qiang¹(), ZHANG Nan^△(), FENG Guangling¹, SUN Weiyi², ZHAO Yuanyuan¹, YANG Shifa¹

1 Henan University of Traditional Chinese Medicine, Zhengzhou 450000, China
2 Department of General Minimally Invasive Surgery, the First Affiliated Hospital of Henan University of Traditional Chinese Medicine

Received:2022-04-01 Revised:2022-09-02 Published:2023-03-15 Online:2023-03-02
Contact: ZHANG Nan E-mail:zqhnzyydx@163.com;znhnzyydx@163.com

郅强, 张楠, 冯光玲, 孙维义, 赵媛媛, 杨世发. 基于PI3K/Akt/mTOR信号通路探讨黄芪多糖对结直肠癌自噬的影响[J]. 天津医药, 2023, 51(3): 240-245.

ZHI Qiang, ZHANG Nan, FENG Guangling, SUN Weiyi, ZHAO Yuanyuan, YANG Shifa. Study on the mechanism of astragalus polysaccharides regulating autophagy of colorectal cancer cells based on PI3K/Akt/mTOR signaling pathway[J]. Tianjin Medical Journal, 2023, 51(3): 240-245.

摘要/Abstract

摘要： Objective To investigate the effect and mechanism of astragalus polysaccharides (APS) on autophagy in colorectal cancer. Methods The effects of various concentrations of APS (0.25 g/L, 0.5 g/L, 0.75 g/L and 1 g/L) on the proliferation of human colorectal cancer HCT-116 cells were detected by CCK-8 test. The effect of APS on the migration of HCT-116 cells was detected by cell scratch assay. The effect of APS on autophagy of colorectal cancer cells was detected by dansylcadaverine staining technique. Effects of APS on autophagy and PI3K/Akt/mTOR signaling pathway related proteins LC3B, p62, p-PI3K, p-Akt, p-mTOR, PI3K, Akt and mTOR in HCT-116 cells and colorectal cancer tumor-bearing nude mice were detected by Western blot assay. The expression of autophagy-related protein p62 in colorectal cancer tumors was detected by immunohistochemistry. Results APS inhibited HCT-116 cell proliferation in a dose-dependent manner (P<0.05). APS could inhibit the proliferation and migration of HCT-116 cells by inducing autophagy (P<0.05). The autophagy inhibitor 3-MA (2 mmol/L) not only attenuated the autophagy induction effect of APS on HCT-116 cells, but also attenuated the inhibitory effect of APS on the proliferation and migration of HCT-116 cells (P<0.05). APS could increase the expression ratio of autophagy-related protein LC3BⅡ/Ⅰ and reduce the expression level of p62 protein in HCT-116 cells and colorectal cancer mice (P<0.05). 3-MA could attenuate the increasing effect of APS on the expression ratio of LC3BⅡ/Ⅰ protein and reduce the inhibitory effect of APS on the expression of p62 protein in HCT-116 cells (P<0.05). APS could reduce the expression ratio of PI3K/Akt/mTOR signaling pathway related proteins p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR in HCT-116 cells and colorectal cancer tumor (P<0.05). The pathway agonist 740Y-P (10 μmol/L) could attenuate the inhibitory effect of APS on expression levels of PI3K/Akt/mTOR signaling pathway-related proteins in HCT-116 cells (P<0.05). Conclusion APS can induce autophagy activation in colorectal cancer tumor tissue and cells, and inhibit cell proliferation and migration of HCT-116 cells by inducing autophagy. The mechanism of action may be related to the inhibition of PI3K/Akt/mTOR signaling pathway.

关键词: 黄芪多糖, 结直肠癌, 自噬, PI3K/Akt/mTOR信号通路

Key words: astragalus polysaccharides, colorectal cancer, autophagy, PI3K/Akt/mTOR signaling pathway

中图分类号:

R285.5

图/表 12

表1 各组HCT-116细胞不同时点增殖活性比较 (n=3,%,$\bar{x}±s$)

Tab.1 Comparison of proliferation activity of HCT-116 cells at different time points between the five groups

组别	12 h	24 h	48 h
control组	100.00±0.00	100.00±0.00	100.00±0.00
0.25 g/L APS组	96.66±1.46	91.27±1.28^a	75.88±2.26^a
0.5 g/L APS组	88.30±4.85^a	80.26±3.45^ab	62.85±5.85^ab
0.75 g/L APS组	81.41±3.61^ab	57.83±2.89^abc	46.36±1.32^abc
1.0 g/L APS组	69.69±5.05^abcd	51.09±1.51^abcd	39.69±1.96^abc
F	34.740^＊	276.200^＊	195.200^＊

图1 各组HCT-116细胞MDC染色比较（×200）

Fig.1 Comparison of MDC staining between the three groups of HCT-116 cells（×200）

图2 各组HCT-116细胞迁移比较（×40）

Fig.2 Comparison of HCT-116 cell migration between the four groups（×40）

表2 各组HCT-116细胞增殖活性和迁移率比较(n=3,%,$\bar{x}±s$)

Tab.2 Comparison of proliferation activity and migration rate of HCT-116 cells between the four groups

组别	细胞增殖活性	细胞迁移率
control组	100.00±0.00	51.62±1.86
APS组	54.20±3.67^a	35.09±1.18^a
3-MA组	95.15±2.20^b	49.02±1.56^b
APS+3-MA组	70.54±2.36^abc	42.70±1.19^abc
F	231.900^＊	74.950^＊

表3 各组HCT-116细胞自噬相关蛋白表达水平比较(n=3,$\bar{x}±s$)

Tab.3 Comparison of autophagy related protein expression levels of HCT-116 cells between the four groups

组别	LC3BⅡ/Ⅰ	p62
control组	1.00±0.00	1.00±0.00
APS组	1.77±0.11^a	0.61±0.07^a
3-MA组	0.57±0.14^ab	1.49±0.22^ab
APS+3-MA组	1.01±0.13^bc	1.23±0.16^b
F	60.520^＊	21.030^＊

图3 各组HCT-116细胞自噬相关蛋白p62及LC3BⅡ/Ⅰ表达

Fig.3 Expression of autophagy related protein p62 and LC3BⅡ/Ⅰ in HCT-116 cells of each group

表4 各组HCT-116细胞PI3K/Akt/mTOR通路相关蛋白相对表达量比较(n=3,$\bar{x}±s$)

Tab.4 Comparison of relative expression levels of PI3K/Akt/mTOR pathway related proteins in HCT-116 cells between the four groups

组别	p-PI3K/PI3K	p-Akt/Akt	p-mTOR/mTOR
control组	1.00±0.00	1.00±0.00	1.00±0.00
APS组	0.70±0.05^a	0.58±0.23^a	0.71±0.06^a
740Y-P组	1.48±0.19^ab	1.50±0.11^ab	1.24±0.10^ab
APS+740Y-P组	1.16±0.11^bc	1.10±0.15^bc	0.92±0.05^bc
F	25.920^＊	19.360^＊	36.060^＊

图4 各组HCT-116细胞PI3K/Akt/mTOR信号通路相关蛋白表达

Fig.4 Expression of PI3K/Akt/mTOR signal pathway related proteins in HCT-116 cells of each group

表5 各组结直肠癌小鼠肿瘤自噬、PI3K/Akt/mTOR通路相关蛋白表达水平比较 (n=3, $\bar{x}$±s)

Tab.5 Comparison of autophagy and PI3K/Akt/mTOR pathway related protein expression levels between the three groups of colorectal cancer mice

组别		LC3BⅡ/Ⅰ		p62		p-PI3K/PI3K
control组		1.00±0.00		1.00±0.00		1.00±0.00
APS L组		1.40±0.06^a		0.75±0.08^a		0.77±0.04^a
APS H组		1.93±0.19^ab		0.52±0.09^ab		0.61±0.05^ab
F		48.580^＊		37.340^＊		73.780^＊
组别	p-Akt/Akt		p-mTOR/mTOR		p62（IOD）
control组	1.00±0.00		1.00±0.00		53 899.86±2 035.99
APS L组	0.76±0.07^a		0.73±0.04^a		36 337.14±1 527.26^a
APS H组	0.58±0.03^ab		0.58±0.02^ab		13 444.72±5 567.52^ab
F	76.990^＊		263.500^＊		98.830^＊

图5 各组结直肠癌小鼠肿瘤LC3BⅡ/Ⅰ和p62蛋白表达

Fig.5 Expression of LC3BⅡ/Ⅰ and p62 proteins in colorectal cancer mice of each group

图6 各组结直肠癌小鼠肿瘤p62蛋白的免疫组化染色比较（×200）

Fig.6 Comparison of p62 protein immunohistochemical staining in colorectal cancer mice between the three groups （×200）

图7 各组结直肠癌小鼠肿瘤PI3K/Akt/mTOR信号通路相关蛋白表达

Fig.7 Expression of PI3K/Akt/mTOR signal pathway related proteins in colorectal cancer mice of each group

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基于PI3K/Akt/mTOR信号通路探讨黄芪多糖对结直肠癌自噬的影响

Study on the mechanism of astragalus polysaccharides regulating autophagy of colorectal cancer cells based on PI3K/Akt/mTOR signaling pathway

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