基于蛋白质组学探讨消癌解毒方调控Nrf2/HMOX1通路促进肝癌细胞铁死亡的作用机制

李文婷; 张淇; 吴勉华; 李黎; 姜泽群; 张羽; 杨卫豪; 费凡

doi:10.14148/j.issn.1672-0482.2023.1179

基于蛋白质组学探讨消癌解毒方调控Nrf2/HMOX1通路促进肝癌细胞铁死亡的作用机制

doi: 10.14148/j.issn.1672-0482.2023.1179

李文婷^{1, 2,},
张淇^{1, 2},
吴勉华^{1, 2, ,},
李黎^{1, 2},
姜泽群^{2, 3},
张羽^{2, 3},
杨卫豪^{1, 2},
费凡^{2, 3}

1.
南京中医药大学第一临床医学院, 江苏南京 210023
2.
江苏省中医药防治肿瘤协同创新中心, 江苏南京 210023
3.
南京中医药大学医学院·整合医学院, 江苏南京 210023

基金项目:

国家自然科学基金青年科学基金项目 81804058

第二届全国名中医传承工作室国中医药办人教函〔2022〕245号

吴勉华全国名老中医药专家传承工作室国中医药人教函〔2022〕75号

国家中医药管理局第七批全国老中医药专家学术经验继承工作项目国中医药人教函〔2022〕76号

吴勉华江苏省名老中医药专家传承工作室苏中医科教〔2021〕7号

国家中医药管理局中医药传承与创新“百千万”人才工程(岐黄工程)岐黄学者项目国中医药人教函〔2018〕284号

国家级大学生创新训练计划项目 202110315012

详细信息

作者简介:
李文婷, 女, 讲师, E-mail: 260729@njucm.edu.cn

通讯作者:
吴勉华, 男, 教授, 主任中医师, 主要从事中医药抗肿瘤作用机制的研究, E-mail: wmh7001@163.com

中图分类号: R285.5
计量
- 文章访问数: 242
- HTML全文浏览量: 31
- PDF下载量: 30
- 被引次数: 0
出版历程
- 收稿日期: 2023-06-30
- 网络出版日期: 2023-12-20
- 发布日期: 2023-12-10

Proteomics-Based Study on the Mechanism of Xiaoai Jiedu Recipe Regulating Nrf2/HMOX1 Pathway to Promote Ferroptosis in Hepatocellular Carcinoma Cells

LI Wen-ting^{1, 2
,},
ZHANG Qi^{1, 2},
WU Mian-hua^{1, 2
, ,},
LI Li^{1, 2},
JIANG Ze-qun^{2, 3},
ZHANG Yu^{2, 3},
YANG Wei-hao^{1, 2},
FEI Fan^{2, 3}

1.
The First School of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing 210023, China
3.
School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China

摘要

摘要: 目的通过蛋白质组学方法探讨消癌解毒方促进肝癌小鼠肿瘤细胞铁死亡, 抑制移植瘤生长的作用机制。方法 C57BL/6J小鼠随机分为模型组、消癌解毒方低剂量组、消癌解毒方高剂量组、消癌解毒方联合铁死亡抑制剂组、铁死亡抑制剂组、顺铂组。构建H22小鼠移植瘤模型, 消癌解毒方低、高剂量组予消癌解毒方灌胃, 剂量分别为(10、20 g·kg^-1·d^-1); 铁死亡抑制剂组予Liproxstatin-1腹腔注射, 剂量为10 mg·kg^-1·d^-1; 顺铂组予顺铂腹腔注射, 剂量为10 mg·kg^-1·d^-1; 消癌解毒方联合铁死亡抑制剂组予消癌解毒方(20 g·kg^-1·d^-1)灌胃以及Liproxstatin-1(10 mg·kg^-1·d^-1)腹腔注射, 模型组灌胃等量生理盐水。连续给药11 d后剥取瘤体计算抑瘤率；HE染色检测病理变化；透射电镜观察线粒体结构变化；流式细胞术检测瘤体组织活性氧(ROS)水平；制备血清以TMT肽段标记结合LC-MS/MS寻找差异蛋白表达谱、应用IPA软件进行分析；生化检测血清铁离子、还原型谷胱甘肽(GSH)及丙二醛(MDA)含量；Western blot法检测核因子E2相关因子2(Nrf2)、血红素加氧酶-1(HMOX1)、胱氨酸/谷氨酸逆向转运蛋白(SLC7A11)和谷胱甘肽过氧化物酶4(GPX4)的蛋白表达水平。结果消癌解毒方低、高剂量及顺铂能抑制瘤体生长, 抑瘤率分别为36.12%、51.63%、57.43%, 铁死亡抑制剂促进了瘤体的生长, 抑瘤率为-45.56%, 消癌解毒方联合铁死亡抑制剂组瘤体较铁死亡抑制剂组缩小, 抑瘤率为18.11%;HE染色显示消癌解毒方高剂量组、顺铂组瘤体组织出现凋亡细胞及大量空泡蓄积; 透射电镜结果显示, 消癌解毒方低、高剂量组出现一定程度的线粒体萎缩及膜密度增加; 流式结果显示: 消癌解毒方干预后ROS水平明显升高(P < 0.01);蛋白质组学检测显示, 消癌解毒方高剂量组与模型组相比差异蛋白共129个, 其中下调蛋白62个, 上调蛋白67个, 差异表达涉及脂质代谢等方面; 生化检测结果显示, 消癌解毒方干预可升高小鼠血清铁离子、MDA含量, 降低GSH含量; Western blot结果显示, 消癌解毒方高剂量组干预后Nrf2、HMOX1蛋白表达水平增高, SLC7A11、GPX4蛋白表达水平降低(P < 0.01)。结论消癌解毒方可调控Nrf2/HMOX1信号通路, 调节氧化应激, 升高脂质过氧化水平, 促进肝癌细胞铁死亡, 可能是其抑制移植瘤生长的作用机制之一。
- 消癌解毒方 /
- Nrf2/HMOX1通路 /
- 氧化应激 /
- 脂质过氧化 /
- 铁死亡
Abstract: OBJECTIVE To investigate the mechanism of Xiaoai Jiedu Recipe in promoting ferroptosis of tumor cells and inhibiting the growth of transplanted tumors in hepatocellular carcinoma mice by proteomics method. METHODS C57BL/6J mice were randomly divided into, model group, low- and high-dose groups of Xiaoai Jiedu Recipe, Xiaoai Jiedu Recipe combined with ferroptosis inhibitor group, ferroptosis inhibitor group and cisplatin group. The H22 mouse transplantation tumor model was constructed and the drug administration interventions were as follows: the low- and high-dose groups of Xiaoai Jiedu Recipe were given Xiaoai Jiedu Recipe by gavage at the doses of 10 and 20 g·kg^-1·d^-1; the ferroptosis inhibitor group was given Liproxstatin-1 by intraperitoneal injection at the dose of 10 mg·kg^-1·d^-1; the cisplatin group was given cisplatin by intraperitoneal injection at the dose of 10 mg·kg^-1·d^-1; the Xiaoai Jiedu Recipe combined with ferroptosis inhibitor group was given a gavage dose of 20 g·kg^-1·d^-1of Xiaoai Jiedu Recipe and an intraperitoneal injection of 10 mg·kg^-1·d^-1 of the ferroptosis inhibitor Liproxstatin-1; the model group was given an equal amount of saline by gavage. The drugs were administered for 11 d continuously. The tumors were stripped to calculate tumor inhibition rate. Pathological changes were observed by hematoxylin-eosin (HE). Mitochondrial structural changes were observed by transmission electron microscopy. Reactive oxygen species (ROS) levels were detected by flow cytometry. Serum was prepared and analysed by TMT peptide labelling combined with LC-MS/MS to find differential protein expression profiles, applying IPA software. Serum iron ions, glutathione (GSH) and malondialdehyde (MDA) levels were measured biochemically. Protein expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HMOX1), cystine/glutamate reverse transporter protein (SLC7A11) and glutathione peroxidase 4 (GPX4) were measured by protein Western blot. RESULTS The tumor growth was inhibited in the low- and high-dose groups and the cisplatin group, with inhibition rates of 36.12%, 51.63% and 57.43%, respectively, while the tumor growth was promoted in the ferroptosis inhibitor group, with an inhibition rate of -45.56%, and the tumor size was reduced in the Xiaoai Jiedu Recipe combined with ferroptosis inhibitor group compared with the ferroptosis inhibitor group, with an inhibition rate of 18.11%. HE staining showed that apoptotic cells and a large number of vacuoles accumulated in the tumor tissues of the high-dose group and the cisplatin group. Transmission electron microscopy showed that the mitochondrial atrophy and membrane density increased to a certain extent in the low and high-dose groups of Xiaoai Jiedu Recipe. Flow cytometry results showed that ROS levels were significantly increased after the intervention of Xiaoai Jiedu Recipe (P < 0.01). Proteomic tests showed that there were 129 differential proteins, including 62 down-regulated proteins and 67 up-regulated proteins, in the high-dose group of Xiaoai Jiedu Recipe compared with the model group, and these differential expressions were involved in lipid metabolism, et al. The results of biochemical tests showed that the intervention of Xiaoai Jiedu Recipe increased the serum iron and MDA levels, and decreased the GSH level in mice. Western blot results showed that the protein expression levels of Nrf2 and HMOX1 increased, and those of SLC7A11 and GPX4 decreased after the intervention of the high-dose group of Xiaoai Jiedu Recipe (P < 0.01). CONCLUSION Xiaoai Jiedu Recipe promotes ferroptosis in hepatocellular carcinoma cells by inducing the Nrf2/HMOX1 signaling pathway, regulating oxidative stress and elevating the level of lipid peroxidation, which may be one of its mechanisms of action in inhibiting the growth of transplanted tumors.
- Xiaoai Jiedu Recipe /
- Nrf2/HMOX1 pathway /
- oxidative stress /
- lipid peroxidation /
- ferroptosis

HTML全文

图 1 各组小鼠瘤体组织HE染色(×200)

注: A.模型组; B.消癌解毒方低剂量组; C.消癌解毒方高剂量组; D.消癌解毒方联合铁死亡抑制剂组; E.铁死亡抑制剂组; F.顺铂组。

Figure 1. HE staining of tumor tissue in each group of mice(×200)

下载: 全尺寸图片幻灯片

图 2 各组小鼠瘤体细胞透射电镜图(×20 000)

注: A.模型组; B.消癌解毒方低剂量组; C.消癌解毒方高剂量组; D.消癌解毒方联合铁死亡抑制剂组; E.铁死亡抑制剂组; F.顺铂组。

Figure 2. Transmission electron micrograph of each group of tumor cells(×20 000)

下载: 全尺寸图片幻灯片

图 3 消癌解毒方对瘤体组织ROS水平的影响(x±s, n=3)

注: A.模型组; B.消癌解毒方低剂量组; C.消癌解毒方高剂量组; D.消癌解毒方联合铁死亡抑制剂组; E.铁死亡抑制剂组; F.顺铂组。与模型组比较, ^{* *}P < 0.01;与铁死亡抑制剂组比较, ^##P < 0.01。

Figure 3. Effect of Xiaoai Jiedu Recipe on the level of ROS in tumor tissue(x±s, n=3)

下载: 全尺寸图片幻灯片

图 4 模型组与消癌解毒方高剂量组血清差异蛋白聚类图

注: G1.模型组; G2.消癌解毒方高剂量组；r1.样本第1次检测结果；r2.样本重复1次后检测结果。n=6。

Figure 4. Serum differential protein clustering in the model group compared to the Xiaoai Jiedu Recipe group

下载: 全尺寸图片幻灯片

图 5 消癌解毒方高剂量组与模型组差异蛋白富集度最高的通路图

Figure 5. The pathway with the highest differential protein enrichment in the high dose group of Xiaoai Jiedu Recipe compared to the model group

下载: 全尺寸图片幻灯片

图 6 消癌解毒方高剂量组与模型组, 差异蛋白富集分析中以HMOX1为中心的蛋白质亚网络图

注: 红色表示用药后上调; 绿色表示用药后下调。颜色深浅代表蛋白质表达量变化的大小；没有颜色的表示虽未捕捉到但有文献支持。

Figure 6. Sub-network map of HMOX1-centred proteins in differential protein enrichment analysis in the high-dose group of Xiaoai Jiedu Recipe compared with the model group

下载: 全尺寸图片幻灯片

图 7 消癌解毒方对各组小鼠血清铁、GSH、MDA含量的影响(x±s, n=6)

注: A.模型组; B.消癌解毒方低剂量组; C.消癌解毒方高剂量组; D.消癌解毒方联合铁死亡抑制剂组; E.铁死亡抑制剂组; F.顺铂组。与模型组比较, ^*P < 0.05, ^{* *}P < 0.01;与铁死亡抑制剂组比较, ^##P < 0.01。

Figure 7. Effect of Xiaoai Jiedu Recipe on serum Fe, GSH, MDA content in each group of mice(x±s, n=6)

下载: 全尺寸图片幻灯片

图 8 各组小鼠瘤体组织Nrf2、HMOX1、SLC7A11、GPX4蛋白表达水平

Figure 8. Protein expression levels of Nrf2, HMOX1, SLC7A11 and GPX4 protein of tumor tissue in each group of mice

下载: 全尺寸图片幻灯片

表 1 消癌解毒方对小鼠移植瘤生长的抑制作用(x±s, n=6)

Table 1. Inhibitory effect of Xiaoai Jiedu Recipe on the growth of transplanted tumors in mice(x±s, n=6)

组别	瘤质量/g	抑瘤率/%
模型组	1.399±0.147	-
消癌解毒方低剂量组	0.893±0.040^**	36.12
消癌解毒方高剂量组	0.677±0.037^**	51.63
消癌解毒方联合铁死亡抑制剂组	1.145±0.037^**	18.11
铁死亡抑制剂组	2.036±0.125^**	-45.56
顺铂组	0.595±0.041^**	57.43
注: 与模型组比较，^{* *}P < 0.01。

下载: 导出CSV

表 2 消癌解毒方高剂量组与模型组差异表达明显的部分蛋白

Table 2. Some proteins differentially expressed in the high-dose group of Xiaoai Jiedu Recipe compared with the model group

基因	全称	P值	FC
HMOX1	Heme oxygenase 1	0.000 1	5.03
AKR1C1	Aldo-keto reductase family 1 member C1	0.010 8	4.12
AKR1D1	3-oxo-5-beta-steroid 4-dehydrogenase	0.001 1	3.03
MSMO1	Methylsterol monooxygenase 1	0.000 9	2.74
ZNF639	Zinc finger protein 639	0.000 2	2.62
AKR1C3	Aldo-keto reductase family 1 member C3	0.000 6	2.60
SCD	Acyl-CoA desaturase	0.002 8	2.32
HMGCS1	Hydroxymethylglutaryl-CoA synthase, cytoplasmic	0.003 3	2.21
ACSS2	Isoform 2 of Acetyl-coenzyme A synthetase, cytoplasmic	0.006 2	2.15
SLC2A6	Solute carrier family 2, facilitated glucose transporter member 6	0.003 9	2.13
FDFT1	Squalene synthase	0.000 5	2.08
UNC84A	SUN domain-containing protein 1	0.000 2	2.00
HAUS4	HAUS augmin-like complex subunit 4	0.004 1	-1.63
TPM2	Isoform 2 of Tropomyosin beta chain	0.011 4	-1.64
KRT10	Keratin, type I cytoskeletal 10	0.009 4	-1.64
EHMT2	Histone-lysine N-methyltransferase EHMT2	0.015 4	-1.73
STEAP4	Metalloreductase STEAP4	0.006 8	-1.74
PZP	Pregnancy zone protein	0.006 3	-1.78
PAIP2	Polyadenylate-binding protein-interacting protein 2	0.015 8	-1.78
MT2A	Metallothionein-2	0.005 1	-1.78
EIF4A1	Eukaryotic initiation factor 4A-I (Fragment)	0.006 2	-2.12
SLC8A1	Sodium/calcium exchanger 1	0.002 6	-3.45
KRT81	Keratin, type Ⅱ cuticular Hb1	0.018 4	-4.07

下载: 导出CSV

参考文献(26)

[1]	中华人民共和国国家卫生健康委员会. 原发性肝癌诊疗指南(2022年版)[J]. 肿瘤综合治疗电子杂志, 2022, 8(2): 16-53. https://www.cnki.com.cn/Article/CJFDTOTAL-XIBU202304002.htm National Health Commission of the People's Republic of China. Guidelines for the diagnosis and treatment of primary hepatic carcinoma(2022 edition)[J]. J Multidiscip Cancer Manag Electron Version, 2022, 8(2): 16-53. https://www.cnki.com.cn/Article/CJFDTOTAL-XIBU202304002.htm
[2]	SIA D, VILLANUEVA A, FRIEDMAN SL, et al. Liver cancer cell of origin, molecular class, and effects on patient prognosis[J]. Gastroenterology, 2017, 152(4): 745-761. doi: 10.1053/j.gastro.2016.11.048
[3]	徐菲, 曾杨丽, 李娟, 等. 中药复方防治肝癌作用机制研究进展[J]. 中国实验方剂学杂志, 2019, 25(24): 196-204. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX201924039.htm XU F, ZENG YL, LI J, et al. Mechanism of traditional Chinese medicine compound in preventing and treating hepatocellular carcinoma[J]. Chin J Exp Tradit Med Formulae, 2019, 25(24): 196-204. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX201924039.htm
[4]	曹毛毛, 陈万青. 中国恶性肿瘤流行情况及防控现状[J]. 中国肿瘤临床, 2019, 46(3): 145-149. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGZL201903010.htm CAO MM, CHEN WQ. Epidemiology of cancer in China and the current status of prevention and control[J]. Chin J Clin Oncol, 2019, 46(3): 145-149. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGZL201903010.htm
[5]	陈海彬, 周红光, 程海波, 等. 消癌解毒方对中晚期恶性肿瘤患者免疫功能的影响[J]. 南京医科大学学报(自然科学版), 2009, 29(9): 1257-1259. https://www.cnki.com.cn/Article/CJFDTOTAL-NJYK200909016.htm CHEN HB, ZHOU HG, CHENG HB, et al. Immune function of Xiaoaijiedufang on patients with advanced malignant tumor[J]. Acta Univ Med Nanjing Nat Sci, 2009, 29(9): 1257-1259. https://www.cnki.com.cn/Article/CJFDTOTAL-NJYK200909016.htm
[6]	周红光, 陈海彬, 吴勉华, 等. 消癌解毒方配合化疗治疗中晚期恶性肿瘤临床疗效观察[J]. 中华中医药杂志, 2010, 25(7): 1140-1143. https://www.cnki.com.cn/Article/CJFDTOTAL-BXYY201007060.htm ZHOU HG, CHEN HB, WU MH, et al. Clinical observation of Xiao'ai Jiedu Prescription combined with chemotherapy on advanced cancer[J]. China J Tradit Chin Med Pharm, 2010, 25(7): 1140-1143. https://www.cnki.com.cn/Article/CJFDTOTAL-BXYY201007060.htm
[7]	QIU WL, WANG ZQ, CHEN R, et al. Xiaoai Jiedu recipe suppresses hepatocellular carcinogenesis through the miR-200b-3p/Notch1 axis[J]. Cancer Manag Res, 2020, 12: 11121-11131.
[8]	WANG YC, XU CH, XU B, et al. Xiaoai Jiedu recipe inhibits proliferation and metastasis of non-small cell lung cancer cells by blocking the P38 mitogen-activated protein kinase (MAPK) pathway[J]. Med Sci Monit, 2019, 25: 7538-7546.
[9]	刘兵, 马英创. 消癌解毒方提取物对乳腺癌细胞增殖活力、细胞周期蛋白、PI3K/AKT通路的调节作用[J]. 中国处方药, 2021, 19(7): 7-9. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGCF202107007.htm LIU B, MA YC. Regulatory effects of Xiaoai Jiedu Decoction extracts on proliferation activity, cyclins and PI3K/AKT pathway of breast cancer cells[J]. J China Prescr Drug, 2021, 19(7): 7-9. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGCF202107007.htm
[10]	沈政洁, 黎思苑, 徐丽贤, 等. 消癌解毒方含药血清增强NK细胞杀伤结肠癌的作用及机制[J]. 中国实验方剂学杂志, 2022, 28(13): 85-91. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX202213011.htm SHEN ZJ, LI SY, XU LX, et al. Xiaoai Jiedu prescription-containing serum enhances lethal effect of NK cells on colon cancer cells[J]. Chin J Exp Tradit Med Formulae, 2022, 28(13): 85-91. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX202213011.htm
[11]	DIXON SJ, LEMBERG KM, LAMPRECHT MR, et al. Ferroptosis: An iron-dependent form of nonapoptotic cell death[J]. Cell, 2012, 149(5): 1060-1072.
[12]	李文婷, 吴勉华, 赵凤鸣, 等. 比较蛋白质组学分析揭示消癌解毒方含药血清作用人肝癌SMMC-7721细胞差异蛋白表达[J]. 中华中医药杂志, 2016, 31(5): 1828-1835. https://www.cnki.com.cn/Article/CJFDTOTAL-BXYY201605065.htm LI WT, WU MH, ZHAO FM, et al. Effects of Xiao'ai Jiedu Formula drug-containing serum on expression of differential proteins in human hepatoma SMMC-7721 cells by comparative proteomic analysis[J]. China J Tradit Chin Med Pharm, 2016, 31(5): 1828-1835. https://www.cnki.com.cn/Article/CJFDTOTAL-BXYY201605065.htm
[13]	TONELLI C, CHIO ⅡC, TUVESON DA. Transcriptional regulation by Nrf2[J]. Antioxid Redox Signal, 2018, 29(17): 1727-1745.
[14]	MA Q. Role of nrf2 in oxidative stress and toxicity[J]. Annu Rev Pharmacol Toxicol, 2013, 53: 401-426.
[15]	RYTER SW. Heme oxgenase-1, a cardinal modulator of regulated cell death and inflammation[J]. Cells, 2021, 10(3): 515.
[16]	SUTTNER DM, DENNERY PA. Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron[J]. FASEB J, 1999, 13(13): 1800-1809.
[17]	GORRINI C, HARRIS IS, MAK TW. Modulation of oxidative stress as an anticancer strategy[J]. Nat Rev Drug Discov, 2013, 12(12): 931-947.
[18]	CHIANG SK, CHEN SE, CHANG LC. A dual role of heme oxygenase-1 in cancer cells[J]. Int J Mol Sci, 2018, 20(1): 39.
[19]	HASSANNIA B, WIERNICKI B, INGOLD I, et al. Nano-targeted induction of dual ferroptotic mechanisms eradicates high-risk neuroblastoma[J]. J Clin Invest, 2018, 128(8): 3341-3355.
[20]	YANG WS, STOCKWELL BR. Ferrotosis: Death by Lipid Peroxidation[J]. Trends Cell Biol, 2016, 26(3): 165-176.
[21]	LIU JY, XIA XJ, HUANG P. xCT: A critical molecule that links cancer metabolism to redox signaling[J]. Mol Ther, 2020, 28(11): 2358-2366.
[22]	KOPPULA P, ZHUANG L, GAN BY. Cystine transporter SLC7A11/xCT in cancer: Ferroptosis, nutrient dependency, and cancer therapy[J]. Protein Cell, 2021, 12(8): 599-620.
[23]	STOCKWELL BR, FRIEDMANN ANGELI JP, BAYIR H, et al. Ferroptosis: A regulated cell death nexus linking metabolism, redox biology, and disease[J]. Cell, 2017, 171(2): 273-285.
[24]	YANG WS, SRIRAMARATNAM R, WELSCH ME, et al. Regulation of ferroptotic cancer cell death by GPX4[J]. Cell, 2014, 156(1/2): 317-331.
[25]	ALIM I, CAULFIELD JT, CHEN YX, et al. Selenium drives a transcriptional adaptive program to block ferroptosis and treat stroke[J]. Cell, 2019, 177(5): 1262-1279. e25.
[26]	SHARMA GN, GUPTA G, SHARMA P. A comprehensive review of free radicals, antioxidants, and their relationship with human ailments[J]. Crit Rev Eukaryot Gene Expr, 2018, 28(2): 139-154.