Study on the Inhibition of Esophageal Carcinoma Development in Mice by Salvia Chinensia Benth Induced Ferroptosis

doi:10.12114/j.issn.1007-9572.2023.0342

Abstract

Abstract:

Background

Esophageal carcinoma is a common malignant tumor of the gastrointestinal tract in China and even globally, with a high incidence and mortality rate. As a traditional Chinese medicine, Salvia chinensia Benth (SJC) has the effects of clearing heat and detoxifying, promoting blood circulation and easing pain in the treatment of esophageal cancer. Pharmacological experimental studies have proved that SJC has anticancer properties and can effectively treat a variety of malignant tumors.

Objective

To explore the effect and mechanism of SJC in the inhibition of carcinoma in situ esophageal cancer development of C57 mice based on ferroptosis.

Methods

Ninety SPF grade C57BL/6 female mice were selected from February 2022 to February 2023 and randomly divided into Control group (n=15), simple 4-Nitroquinoline N-oxide (4NQO) -induced cancer group (4NQO group, n=25), 4NQO+ low-dose SJC group[4NQO/SJC (91 mg) group, n=25]and 4NQO+high dose SJC group[4NQO/SJC (182 mg) group, n=25]. The preparation of in situ model of esophageal cancer in C57 mice was carried out using 4NQO induction. The activities of the mice were observed, their mental state, food and water intake were recorded, and the body mass of the mice was measured and recorded at 8-week intervals. Hematoxylin-eosin (HE) staining and pathological analysis of esophageal tissue were performed after 32 weeks. The contents of Fe²⁺, glutathione (GSH) and malondialdehyde (MDA) in esophageal tissues were determined, and the expression levels of nuclear receptor coactivator 4 (NCOA4) and glutathione peroxidase 4 (GPX4) in esophageal tissues of mice were detected by western blot. Kaplan-Meier method was used to plot the survival curves of mice, and Breslow test was used to compare the survival curves.

Results

The body mass of mice in 4NQO group, 4NQO/SJC (91 mg) group and 4NQO/SJC (182 mg) group at 8, 16, 24 and 32 weeks of modeling was lower than that in Control group. The body mass of 4NQO/SJC (91 mg) group and 4NQO/SJC (182 mg) group at 32 weeks was higher than that of 4NQO group (P<0.05). The results of Breslow test showed that there was significant difference in the survival curves of mice in the four groups (χ²=9.907, P=0.019). The results of HE staining showed that esophageal epithelial tissue of mice in 4NQO group showed abnormal proliferation, disordered cell arrangement, and abnormal pathological changes such as keratinized beads. Compared with 4NQO group, the esophageal epithelial histopathological changes in 4NQO/SJC (91 mg) and 4NQO/SJC (182 mg) groups were significantly improved. Fe²⁺ and MDA in 4NQO, 4NQO/SJC (91 mg) and 4NQO/SJC (182 mg) groups were lower than those in Control group, and GSH was higher than that in Control group (P<0.05). Fe²⁺ and MDA in 4NQO/SJC (91 mg) and 4NQO/SJC (182 mg) groups were higher than those in 4NQO group, and GSH was lower than that in 4NQO group (P<0.05). Fe²⁺ and MDA in 4NQO/SJC (182 mg) group were higher than those in 4NQO/SJC (91 mg) group, and GSH in 4NQO/SJC (91 mg) group was lower than that in 4NQO/SJC (91 mg) group (P<0.05). NCOA4 in 4NQO group was lower than that in Control group, 4NQO/SJC (91 mg) group and 4NQO/SJC (182 mg) group, and GPX4 was higher than that in Control group, 4NQO/SJC (91 mg) group and 4NQO/SJC (182 mg) group (P<0.05). The GPX4 of 4NQO/SJC (91 mg) group and 4NQO/SJC (182 mg) group was higher than that of Control group (P<0.05) .

Conclusion

It is proved that SJC can interfere with the development of esophageal cancer development by a mechanism that may be related to NCOA4-mediated ferritin phagocytosis.

Key words: Esophageal carcinoma, Ferroptosis, Salvia chinensia Benth, Induction of carcinoma in situ, Glutathione peroxidase 4, Nuclear receptor coactivator 4

摘要：

背景

食管癌是一种在中国乃至全球范围内常见的消化道恶性肿瘤，具有很高的发病率和致死率。石见穿（SJC）作为传统中药，其清热解毒、活血镇痛之功常用于食管癌的治疗中。药理实验研究证明，SJC具有抗癌性，可有效治疗多种恶性肿瘤。

目的

基于铁死亡探讨SJC抑制C57小鼠食管原位癌发生发展的作用和机制。

方法

2022年2月—2023年2月选取SPF级C57BL/6雌鼠90只，随机分为对照组（Control组，n=15）、单纯4NQO诱癌组（4NQO组，n=25）、4-硝基喹啉N-氧化物（4NQO）+SJC低剂量组[4NQO/SJC（91 mg）组，n=25]和4NQO+SJC高剂量组[4NQO/SJC（182 mg）组，n=25]。采用4NQO诱导的方式进行C57小鼠食管癌原位模型的制备。观察小鼠活动情况，记录其精神状况和进食饮水情况，每隔8周分组测量小鼠体质量并进行记录。32周后进行食管组织苏木素-伊红（HE）染色和病理学分析。测定食管组织Fe²⁺、谷胱甘肽（GSH）和丙二醛（MDA）含量，采用蛋白质印迹法检测小鼠食管组织中核受体共激活因子4（NCOA4），谷胱甘肽过氧化物酶4（GPX4）蛋白质的表达水平。采用Kaplan-Meier法绘制小鼠的生存曲线，生存曲线的比较采用Breslow检验。

结果

造模8、16、24、32周4NQO组、4NQO/SJC（91 mg）组、4NQO/SJC（182 mg）组小鼠体质量低于Control组，32周4NQO/SJC（91 mg）组、4NQO/SJC（182 mg）组小鼠体质量高于4NQO组（P<0.05）。Breslow检验结果显示，4组小鼠生存曲线比较，差异有统计学意义（χ²=9.907，P=0.019）。HE染色结果可见，4NQO组小鼠食管上皮组织呈现异常增生，细胞排列紊乱，出现角化珠等异常病理改变；与4NQO组比较，4NQO/SJC（91 mg）组和4NQO/SJC（182 mg）组食管上皮组织病理学改变明显改善。4NQO组、4NQO/SJC（91 mg）组、4NQO/SJC（182 mg）组Fe²⁺、MDA均低于Control组，GSH均高于Control组（P<0.05）；4NQO/SJC（91 mg）组、4NQO/SJC（182 mg）组Fe²⁺、MDA均高于4NQO组，GSH均低于4NQO组（P<0.05）；4NQO/SJC（182 mg）组Fe²⁺、MDA均高于4NQO/SJC（91 mg）组，GSH均低于4NQO/SJC（91 mg）组（P<0.05）。4NQO组NCOA4低于Control组、4NQO/SJC（91 mg）组、4NQO/SJC（182 mg）组，GPX4高于Control组、4NQO/SJC（91 mg）组、4NQO/SJC（182 mg）组（P<0.05）；4NQO/SJC（91 mg）组、4NQO/SJC（182 mg）组GPX4高于Control组（P<0.05）。

结论

SJC可干预食管癌的发生发展，其机制可能与NCOA4介导的铁蛋白吞噬作用相关。

关键词: 食管癌, 铁死亡, 石见穿, 原位癌诱导模型, 谷胱甘肽过氧化物酶4, 核受体共激活因子4

CLC Number:

R 735.1

LIN Xinrong,JIA Lei,LI Lifeng, et al. Study on the Inhibition of Esophageal Carcinoma Development in Mice by Salvia Chinensia Benth Induced Ferroptosis[J]. Chinese General Practice, 2024, 27(30): 3784-3789. DOI: 10.12114/j.issn.1007-9572.2023.0342.
林鑫荣,贾蕾,李丽峰等. 石见穿诱导铁死亡抑制小鼠食管癌发生发展的研究[J]. 中国全科医学, 2024, 27(30): 3784-3789. DOI: 10.12114/j.issn.1007-9572.2023.0342.

Figures/Tables 7

References 19

[1]	LIU C Q, MA Y L, QIN Q，et al. Epidemiology of esophageal cancer in 2020 and projections to 2030 and 2040[J]. Thorac Cancer，2022,14:3-11. DOI：10.1111/1759-7714.14745.
[2]	UHLENHOPP D J, THEN E O, SUNKARA T，et al. Epidemiology of esophageal cancer：update in global trends，etiology and risk factors[J]. Clin J Gastroenterol，2020,13(6):1010-1021. DOI：10.1007/s12328-020-01237-x.
[3]	刘媛，钱荣康，钱荣华. 石见穿及其提取物抗肿瘤的研究进展[J]. 现代中西医结合杂志，2018,27(30):3417-3420. DOI：10.3969/j.issn.1008-8849.2018.30.034.
[4]	SHI L, LIU Y Q, LI M H，et al. Emerging roles of ferroptosis in the tumor immune landscape：from danger signals to anti-tumor immunity[J]. Febs J，2022,289(13):3655-3665. DOI：10.1111/febs.16034.
[5]	HASSANNIA B, VANDENABEELE P, VANDEN BERGHE T. Targeting ferroptosis to iron out cancer[J]. Cancer Cell，2019,35(6):830-849. DOI：10.1016/j.ccell.2019.04.002.
[6]	XU G X, WANG H, LI X L，et al. Recent progress on targeting ferroptosis for cancer therapy[J]. Biochem Pharmacol，2021,190:114584. DOI：10.1016/j.bcp.2021.114584.
[7]	吴忠冰. 基于"甘润濡养"法探讨启膈方在食管癌发生和转移中的作用[D]. 石家庄：河北医科大学，2022.
[8]	李麦嘉. 酸枣仁汤对间氯苯哌嗪诱导焦虑模型小鼠的干预作用及机制研究[D]. 济南：山东中医药大学，2022.
[9]	WANG K N, HU Y, HAN L L，et al. Salvia chinensis benth inhibits triple-negative breast cancer progression by inducing the DNA damage pathway[J]. Front Oncol，2022,12:882784. DOI：10.3389/fonc.2022.882784.
[10]	WANG N, TAN H Y, CHAN Y T，et al. Identification of WT1 as determinant of heptatocellular carcinoma and its inhibition by Chinese herbal medicine Salvia chinensis Benth and its active ingredient protocatechualdehyde[J]. Oncotarget，2017,8(62):105848-105859. DOI：10.18632/oncotarget.22406.
[11]	梁伟，王松坡. 石见穿的药用成份及在抗肿瘤方面的研究[J]. 现代肿瘤医学，2014,22(10):2492-2494. DOI：10.3969/j.issn.1672-4992.2014.10.74.
[12]	JIA L, LIN X R, GUO W Y，et al. Salvia chinensia Benth induces autophagy in esophageal cancer cells via AMPK/ULK1 signaling pathway[J]. Front Pharmacol，2022,13:995344. DOI：10.3389/fphar.2022.995344.
[13]	ZHANG J J, DU J, KONG N，et al. Mechanisms and pharmacological applications of ferroptosis：a narrative review[J]. Ann Transl Med，2021,9(19):1503. DOI：10.21037/atm-21-1595.
[14]	JIANG X J, STOCKWELL B R, CONRAD M. Ferroptosis：mechanisms，biology and role in disease[J]. Nat Rev Mol Cell Biol，2021,22(4):266-282. DOI：10.1038/s41580-020-00324-8.
[15]	WANG D. Progress in the study of ferroptosis in cancer treatment：state-of-the-Art[J]. Chem Biol Interact，2023,371:110348. DOI：10.1016/j.cbi.2023.110348.
[16]	LI D X, ZHANG M L, CHAO H T. Significance of glutathione peroxidase 4 and intracellular iron level in ovarian cancer cells-utilization of ferroptosis mechanism[J]. Inflamm Res，2021,70(10/11/12):1177-1189. DOI：10.1007/s00011-021-01495-6.
[17]	CHEN X, KANG R, KROEMER G，et al. Ferroptosis in infection，inflammation，and immunity[J]. J Exp Med，2021,218(6):e20210518. DOI：10.1084/jem.20210518.
[18]	GAO M H, MONIAN P, PAN Q H，et al. Ferroptosis is an autophagic cell death process[J]. Cell Res，2016,26(9):1021-1032. DOI：10.1038/cr.2016.95.
[19]	XIA X J, FAN X P, ZHAO M Y，et al. The relationship between ferroptosis and tumors：a novel landscape for therapeutic approach[J]. Curr Gene Ther，2019,19(2):117-124. DOI：10.2174/1566523219666190628152137.

分组	只数	0周	8周	16周^a	24周^a	32周^a
Control组	15	15.47±0.49	17.24±0.53	19.06±0.75	23.12±0.94	29.44±1.91
4NQO组	25	15.56±0.43	16.90±0.35^b	16.08±3.37^b	14.67±4.93^b	12.13±6.76^b
4NQO/SJC（91 mg）组	25	15.46±0.56	16.77±0.44^b	15.58±3.28^b	15.50±3.53^b	16.28±5.86^bc
4NQO/SJC（182 mg）组	25	15.49±0.79	16.66±0.69^b	14.72±4.46^b	15.65±3.62^b	17.15±4.35^bc
F值		0.116	4.269	5.228	18.699	33.228
P值		0.951	0.007	0.002	<0.001	<0.001

分组	只数	0周	8周	16周^a	24周^a	32周^a
Control组	15	15.47±0.49	17.24±0.53	19.06±0.75	23.12±0.94	29.44±1.91
4NQO组	25	15.56±0.43	16.90±0.35^b	16.08±3.37^b	14.67±4.93^b	12.13±6.76^b
4NQO/SJC（91 mg）组	25	15.46±0.56	16.77±0.44^b	15.58±3.28^b	15.50±3.53^b	16.28±5.86^bc
4NQO/SJC（182 mg）组	25	15.49±0.79	16.66±0.69^b	14.72±4.46^b	15.65±3.62^b	17.15±4.35^bc
F值		0.116	4.269	5.228	18.699	33.228
P值		0.951	0.007	0.002	<0.001	<0.001

分组	只数	Fe²⁺	GSH	MDA
Control组	15	1.34±0.06	2.38±0.06	0.36±0.04
4NQO组	14	0.49±0.10^a	8.35±0.08^a	0.18±0.01^a
4NQO/SJC（91 mg）组	19	1.08±0.05^ab	6.11±0.22^ab	0.28±0.02^ab
4NQO/SJC（182 mg）组	20	1.21±0.04^abc	2.90±0.23^abc	0.32±0.04^b
F值		99.246	384.764	19.491
P值		<0.001	<0.001	<0.001

分组	只数	Fe²⁺	GSH	MDA
Control组	15	1.34±0.06	2.38±0.06	0.36±0.04
4NQO组	14	0.49±0.10^a	8.35±0.08^a	0.18±0.01^a
4NQO/SJC（91 mg）组	19	1.08±0.05^ab	6.11±0.22^ab	0.28±0.02^ab
4NQO/SJC（182 mg）组	20	1.21±0.04^abc	2.90±0.23^abc	0.32±0.04^b
F值		99.246	384.764	19.491
P值		<0.001	<0.001	<0.001

分组	只数	NCOA4	GPX4
Control组	15	1.00±0.14	1.00±0.11
4NQO组	14	0.42±0.22^a	1.91±0.26^a
4NQO/SJC（91 mg）组	19	1.05±0.03^b	1.56±0.10^ab
4NQO/SJC（182 mg）组	20	1.22±0.19^b	1.33±0.15^ab
F值		13.735	20.289
P值		0.002	<0.001