药学学报

Gene name	Ligand name	Binding energy/kcal·mol^-1
ATF4	PO4^3-	-3.90
Mn²⁺	-1.20
ART	-7.60
PARP1	Talazoparib	-14.30
ART	-6.90
DDIT3	ART	-6.50

Gene name	Ligand name	Binding energy/kcal·mol^-1
ATF4	PO4^3-	-3.90
Mn²⁺	-1.20
ART	-7.60
PARP1	Talazoparib	-14.30
ART	-6.90
DDIT3	ART	-6.50

基于转录组测序和网络药理学探究青蒿琥酯干预U87和U251脑胶质瘤细胞的药效差异机制

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李涛 ¹ , 毛霞 ²^,^* , 张彦琼 ² , 林娜 ² , Takashi SATO ³ , Koji MIZUNO ³ , Katsuki OKUYAMA ³ , 黄丰 ¹^,^*

药学学报 | 专题报道: 基于整合药理学的中医药现代化研究 2023,58(6): 1475-1483

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药学学报 |专题报道: 基于整合药理学的中医药现代化研究 2023 , 58 (6) : 1475 -1483

基于转录组测序和网络药理学探究青蒿琥酯干预U87和U251脑胶质瘤细胞的药效差异机制

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李涛¹, 毛霞²^,^*, 张彦琼², 林娜², Takashi SATO³, Koji MIZUNO³, Katsuki OKUYAMA³, 黄丰¹^,^*

作者信息

1.云南中医药大学中药学院, 云南昆明 650500

2.中国中医科学院中药研究所, 北京 100700

3.东京药科大学生物化学系, 东京都八王子市 192-0392

通讯作者:

*毛霞, Tel: 86-10-64014411-2869, E-mail: xmao@icmm.ac.cn;

黄丰, Tel: 86-871-67429415, E-mail: 14030@ynutcm.edu.cn

Exploring the potential mechanism of artesunate in intervening with U87 cells and U251 cells with distinct therapeutic effects on the basis of transcriptome sequencing and network pharmacology

Tao LI¹, Xia MAO²^,^*, Yan-qiong ZHANG², Na LIN², Takashi SATO³, Koji MIZUNO³, Katsuki OKUYAMA³, Feng HUANG¹^,^*

Affiliations

1. School of Chinese Materia Medica, Yunnan University of Chinese Medicine, Kunming 650500, China

2. Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

3. Department of Biochemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan

出版时间: 2023-06-12 doi: 10.16438/j.0513-4870.2022-0821

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摘要

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青蒿琥酯具有干预脑胶质瘤的潜能, 但其药理作用机制尚不明确。本研究首先基于U87和U251两种人源脑胶质瘤细胞, 探究青蒿琥酯对细胞活性、增殖和凋亡的影响, 发现青蒿琥酯对U87细胞活性和增殖能力的抑制作用强于对U251细胞; Hoechst和TUNEL细胞凋亡染色实验也发现, 青蒿琥酯可以显著促进U87细胞的凋亡(P < 0.05), 而仅大剂量的青蒿琥酯可促进U251细胞的凋亡(P < 0.01)。接着, 利用青蒿琥酯处理前后的U87和U251细胞裂解液, 通过转录组测序检测和差异数据分析, 分别获得青蒿琥酯敏感性和非敏感性细胞株的差异基因集以及青蒿琥酯效应相关基因集, 旨在挖掘青蒿琥酯对两类细胞敏感性不一的分子机制。通过构建上述各对比组差异基因互作网络和计算网络拓扑特征值, 筛选药效相关的关键网络靶标; 生物功能富集分析结果表明, 上述关键网络靶标显著富集于多条与脑胶质瘤病理环节相关的信号通路, 其中, 与细胞凋亡相关的转录激活因子4 (ATF4)-DNA损伤诱导转录因子3 (DDIT3)-聚腺苷二磷酸核糖聚合酶1 (PARP1) 信号轴的富集显著性最高。分子对接结果也表明, 青蒿琥酯与ATF4和DDIT3均具有较好的结合能力。本研究初步揭示了青蒿琥酯干预U87脑胶质瘤细胞的潜能与其抑制肿瘤细胞内异常活化的ATF4-DDIT3-PARP1信号轴, 进而诱导细胞凋亡有关; 同时也表明了PARP1可能是U251细胞对青蒿琥酯产生耐药的重要靶标。相关结果为青蒿琥酯治疗脑胶质瘤, 以及解决脑胶质瘤治疗药物耐药的现状提供了新的实验依据。

关键词

青蒿琥酯 / 脑胶质瘤 / 转录组测序 / 网络药理学 / 药理机制

Abstract

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Artesunate possesses the potential of intervening with glioma, however, its pharmacological mechanisms remain unclarified. Firstly, the effects of artesunate on cell activity, proliferation and apoptosis of U87 and U251 human glioma cells were explored. It was found that artesunate exerted stronger inhibitory effects on the activity and proliferation of U87 cells than U251 cells. It could significantly promote apoptosis in U87 cells (P < 0.05), while only high dose of artesunate can promote that of U251 cells (P < 0.01), detected by Hoechst and TUNEL cell apoptosis staining. Further, the differential expression gene sets between artesunate-sensitive and non-sensitive cell line, as well the therapeutic effects-related genes of artesunate were obtained through transcriptome sequencing and differential data analysis by using the lysates of U87 and U251 cells before and after artesunate treatment, aiming to explore the molecular mechanism of distinct artesunate sensitivity to two types of cells. Then, key putative targets that related to therapeutic effects were screened by constructing the interaction network of differential genes of three above comparison groups, and calculating their topological characteristics. Pathway enrichment analysis showed that those key putative targets were significantly enriched in several signaling pathways that were closely associated with the main pathological changes of glioma, among which apoptosis-related activating transcription factor 4 (ATF4)-DNA damage induced transcript 3 (DDIT3)- polyadenosine diphosphate ribose polymerase 1 (PARP1) signaling axis was the most enriched in. Molecular docking indicated that artesunate had fine binding affinities with ATF4 and DDIT3. Above all, this study preliminarily revealed that ATF4-DDIT3-PARP1 signaling axis is the target pathway of artesunate intervening with U87 glioma cells, and PARP1 may be an important gene for U251 cells to develop resistance to artesunate. Our results not only provide fundamental experimental evidence for artesunate as a potential therapeutic drug in glioma treatment, but shed light into overcoming drug resistance in its clinical therapy.

Key words

artesunate / glioma / transcriptome sequencing / network pharmacology / pharmacological mechanism

引用本文

李涛, 毛霞, 张彦琼, 林娜, Takashi SATO, Koji MIZUNO, Katsuki OKUYAMA, 黄丰. 基于转录组测序和网络药理学探究青蒿琥酯干预U87和U251脑胶质瘤细胞的药效差异机制. 药学学报, 2023 , 58 (6) : 1475 -1483 . DOI: 10.16438/j.0513-4870.2022-0821

Tao LI, Xia MAO, Yan-qiong ZHANG, Na LIN, Takashi SATO, Koji MIZUNO, Katsuki OKUYAMA, Feng HUANG. Exploring the potential mechanism of artesunate in intervening with U87 cells and U251 cells with distinct therapeutic effects on the basis of transcriptome sequencing and network pharmacology[J]. Acta Pharmaceutica Sinica, 2023 , 58 (6) : 1475 -1483 . DOI: 10.16438/j.0513-4870.2022-0821

正文

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根据国际癌症研究机构(International Agency for Research on Cancer, IARC) 的最新统计, 全世界每年约有10万人被诊断为弥漫性胶质瘤^[1], 占所有新诊断癌症的2%, 有较高的死亡率^[2]。其中, 胶质母细胞瘤(glioblastoma, GBM) 最为致命, 总生存期中位数仅为14~17个月^[3]。目前, 脑胶质瘤的治疗方案主要以手术切除联合放疗化疗的综合治疗为主。由于其较强的侵袭性, 肿瘤在大脑中迅速生长和扩散并入侵周围组织, 外科手术通常无法完全切除患者病灶, 且肿瘤细胞还会对当前的化疗一线药物替莫唑胺(temozolomide, TMZ) 产生耐药^{[4, 5]}, 使得多数患者在治疗过程中对TMZ敏感度降低^[6], 无法满足临床治疗需求。因此, 寻找治疗脑胶质瘤的新型药物迫在眉睫。

青蒿琥酯(artesunate, ART) 是具有倍半萜结构的青蒿素衍生物, 源自菊科蒿属植物黄花蒿(Artemisia annua Linn.), 除了具有抗疟活性^[7]外, 还具有显著的抗肿瘤、调节免疫炎症和抗纤维化等药理活性^[8-10]。现有药理研究表明, 青蒿琥酯具有良好的抗脑胶质瘤潜能, 但其潜在的药理机制并未被完全阐明。有学者表明, ART可促进GBM细胞衰老, 以抑制其增殖^[11]以及通过影响细胞力学特性减弱GBM细胞的迁移和侵袭^[12]; 研究发现, ART和TMZ均可通过诱导细胞凋亡抑制裸鼠皮下肿瘤生长, 但在TMZ应用72 h内联合施用ART效果最好^[13]; 另有研究者通过转铁蛋白修饰的青蒿琥酯纳米脂质体靶向治疗胶质瘤后发现, ART在体内外对胶质瘤均具有良好的疗效^[14]。此外, 大量研究报道脑胶质瘤的发生和恶性进展均与基因表达调控密切相关^[15]。本研究拟基于青蒿琥酯对U87和U251两类脑胶质瘤细胞的体外活性、增殖和凋亡实验, 结合给药前后脑胶质瘤细胞转录组测序(RNA-seq), 构建生物分子互作网络, 探索ART干预两种人源脑胶质瘤细胞的潜在药理机制, 并进行分子对接验证ART与目标靶标的结合能力, 旨在为ART治疗脑胶质瘤提供新的科学依据。

材料与方法

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药品与试剂 人脑胶质瘤细胞U87 (货号: CL-0238) 和U251 (货号: CL-0237) 购自武汉普诺赛生命科技有限公司; ART (货号: A3731, 美国Sigma公司); Cell Titer-Blue^®试剂盒(货号: G8080, 美国Promega公司); 磷酸盐缓冲液(PBS) 粉末(货号: AR0030, 博士德生物工程有限公司); 磷酸盐缓冲液(货号: SH30256.01, 美国Cytiva公司); DMEM培养基(货号: 2186784)、胎牛血清(fetal bovine serum, FBS; 货号: 16000-044)、胰酶(0.25% trypsin-EDTA; 货号: 25200-056) 购自美国Gibco公司; 双抗(penicillin streptomycin solution, PS; 货号: SV30010, 美国Hyclone公司); Hoechst 33258荧光染料(货号: B8030)、TUNEL细胞凋亡试剂盒(货号: T2190)、抗荧光衰减封片剂(货号: S2110) 购自北京索莱宝科技有限公司; 多聚甲醛(货号: G1101, 武汉赛维尔生物科技有限公司)。

Cell Titer-Blue检测细胞活性与增殖 将U87和U251细胞置于5%二氧化碳培养箱中, 于37 ℃下用DMEM培养基培养, 并于对数生长期传代备用。分别取两种细胞(8×10³个/孔) 接种于96孔板, 将实验分为空白组、对照组和实验组。

Cell Titer-Blue检测ART对细胞活性的影响细胞培养24 h后, 实验组加入200 μL含药培养基(分别含ART 0.000 018、0.000 18、0.001 8、0.018、0.18、1.8、18和180 μmol·L^-1), 每组设置6个复孔; 培养24 h后, 加入Cell Titer-Blue试剂20 μL, 孵育2 h后于560 nm处测定吸收度(A) 值, 摸索药物的半数抑制浓度(IC₅₀)。

Cell Titer-Blue检测ART对细胞增殖的影响细胞培养24 h后, U87细胞实验组加入200 μL含药培养基(分别含ART 0.18、1.8和18 μmol·L^-1), 并在培养3、6、10、20和24 h后于560 nm处测定A值; U251细胞实验组加入200 μL含药培养基(分别含ART 1.8、18和180 μmol·L^-1), 并在培养3、6、10、20和24 h后于560 nm处测定A值。

Hoechst染色和TUNEL染色检测细胞凋亡 取对数生长期的U87细胞, 分别加入13.06、26.17 (IC₅₀)、52.34 μmol·L^-1 ART和100 μmol·L^-1 TMZ; 同时取对数生长期的U251细胞, 分别加入90、180、360 μmol·L^-1 ART和250 μmol·L^-1 TMZ。培养24 h后, 进行细胞爬片, 加4%多聚甲醛于室温下固定后, 分别进行Hoechst染色和TUNEL染色, 最后用抗荧光衰减封片剂封片后于荧光显微镜下观察。

转录组测序与网络药理学分析 基于ART作用24 h前后U87 (26.17 μmol·L^-1 ART) 和U251 (180 μmol·L^-1 ART) 脑胶质瘤细胞裂解液, 3 000 r·min^-1, 15 min离心后, 按常规流程进行总RNA提取。质检合格后, 进一步纯化并反转录得双链cDNA, 将其作为模板进行PCR扩增, 构建cDNA文库。高通量测序在Illumina NovaSeq 6000平台上进行, 委托北京致美伊诺生物科技有限公司(中国北京) 完成。

使用fastp软件对原始下机数据进行清理和过滤, 以获得高质量的序列(Clean Reads); 用Hisat2 2.1.0软件将Clean Reads与参考基因进行比对(参考基因组版本为GRCh 38.91); Stringtie 1.3.3b软件对比对结果进行转录组表达定量; 运用TMM (trimmed mean of M values) 算法进行归一化, 最后计算每个基因的FPKM (fragments per kilobase million) 值, 使用edgeR 3.30.3软件包对样本间/组间基因表达进行差异分析。

蛋白质-蛋白质相互作用 (PPI) 网络构建与关键网络靶点筛选 通过上述转录组测序分别获得ART敏感性和非敏感性细胞株的差异基因集以及ART效应相关基因集(U87 vs U251、U87+ART vs U87和U251+ART vs U25)。根据P < 0.05、∣log₂FC∣ > 2.1筛选U87 vs U251间差异基因数; P < 0.05、∣log₂FC∣ > 1.5筛选U87+ART vs U87间差异基因数; P < 0.05、∣log₂FC∣ > 1.0筛选U251+ART vs U251间差异基因数。将上述3组差异基因导入STRING 10.0软件构建PPI网络, 分别获得“U87和U251细胞差异基因”、“ART干预U87细胞效应基因”和“ART干预U251细胞效应基因”的互作网络; 运用Cyto Hubba v 0.1计算上述PPI网络中各节点的网络拓扑特征值(连接度、介度、紧密度和最大集团中心度), 筛选四者均大于相应中位数的节点作为ART敏感性和非敏感性细胞株的差异基因集、ART干预U87细胞效应相关基因集和ART干预U251细胞效应相关基因集中的关键网络靶点。

关键网络靶点的通路富集分析 使用DAVID 6.8对上述关键网络靶标进行基因本体(GO, http://geneontology.org/, last updated on May 16, 2022) 生物过程和京都基因与基因组百科全书(KEGG, http://www.genome.ad.jp/kegg/, last updated on May 21, 2021) 信号通路富集分析(P < 0.05), 探究关键网络靶标参与的生物过程, 并筛选ART干预脑胶质瘤相关的信号通路。

分子对接 从PDB数据库获取转录激活因子4 (activating transcription factor 4, ATF4, ID: 4UT3)、DNA损伤诱导转录因子3 (DNA damage induced transcript 3, DDIT3, ID: 6BJN) 和聚腺苷二磷酸核糖聚合酶1 (polyadenosine diphosphate ribose polymerase 1, PARP1, ID: 7KK3) 3种蛋白质的pdb格式文件, Pubchem数据库中下载ART 3D Conformer SDF格式文件, 并利用Open Babel v3.1.1进行文件格式转换; Discovery Studio 4.5对上述3个蛋白进行前处理后, 使用AutoDock v4.2.6和AutoDock Vina v1.1.2进行分子对接虚拟计算。Pymol 2.4.1可对结果进行可视化。

统计学分析 应用GraphPad Prism 8.0软件进行统计分析。数据用均值±标准差表示, 多组间比较采用单因素方差分析。P < 0.05、P < 0.01和P < 0.001均代表组间差异具有统计意义。

结果

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1 ART对U87细胞活性和增殖能力的抑制作用强于U251细胞

细胞活性和增殖实验结果显示(图 1), ART作用24 h后, 对U87脑胶质瘤细胞的IC₅₀为26.17 μmol·L^-1。与对照组相比, 18 μmol·L^-1 ART自给药3 h起至24 h间, 可显著抑制U87细胞增殖(P < 0.001); 而ART对U251脑胶质瘤细胞的IC₅₀高于最高给药剂量180 μmol·L^-1, 且仅180 μmol·L^-1 ART作用10 h后, 对U251细胞的增殖有影响(P < 0.001)。

2 ART可显著促进U87脑胶质瘤细胞凋亡, 而仅大剂量的ART可促进U251脑胶质瘤细胞的凋亡

Hoechst和TUNEL染色结果显示, 在U87脑胶质瘤细胞中, 3种不同浓度的ART [13.06、26.17 (IC₅₀) 和52.34 μmol·L^-1] 和100 μmol·L^-1 TMZ均可显著诱导细胞凋亡(P < 0.01), 其中, 26.17和52.34 μmol·L^-1 ART诱导效果优于13.06 μmol·L^-1 ART, 26.17 μmol·L^-1 ART诱导效果与100 μmol·L^-1 TMZ相近, 52.34 μmol·L^-1的ART诱导效果优于100 μmol·L^-1 TMZ, 即ART呈浓度依赖性诱导U87细胞凋亡; 在U251脑胶质瘤细胞中, 仅较高浓度的ART (90、180和360 μmol·L^-1) 和250 μmol·L^-1 TMZ方可诱导细胞凋亡(P < 0.01), 其中, 90和180 μmol·L^-1 ART诱导效果与250 μmol·L^-1高浓度TMZ相近, 360 μmol·L^-1 ART诱导效果优于250 μmol·L^-1 TMZ。进一步证实ART和TMZ对U87细胞敏感, 而对U251细胞不敏感(图 2)。

3 ART可能通过调节细胞凋亡相关ATF4-DDIT3-PARP1信号轴而发挥干预U87脑胶质瘤细胞的作用

3.1 ART干预U87脑胶质瘤细胞的效应差异基因明显多于其对U251的效应差异基因

上述细胞体外活性、增殖和凋亡实验结果表明, ART对U87和U251细胞的敏感性存在差异。由此, 本研究进一步采用给药前后的细胞进行转录组测序, 试图挖掘其产生敏感性差异的分子机制。利用ART处理前后的U87和U251细胞裂解液, 通过转录组测序检测和差异数据分析(对比组: U87 vs U251; U87+ART vs U87; U251+ART vs U251), 分别获得ART敏感性和非敏感性细胞株的差异基因集以及ART效应相关基因集。根据P值和log₂FC筛选相应的差异基因数, 结果显示, U87 vs U251中共有1 946个差异基因(1 107个上调基因和839个下调基因); U87+ART vs U87中共有1 858个差异基因(538个上调基因和1 320个下调基因); U251+ART vs U251中共有269个差异基因(181个上调基因和88个下调基因)。由此可见, U87细胞在ART干预前后的效应差异基因数明显多于U251细胞, 这与ART对U87细胞药效敏感性高于U251细胞的体外实验结果相一致。

3.2 细胞凋亡相关ATF4-DDIT3-PARP1信号轴可能是ART干预U87脑胶质瘤细胞的作用靶点之一

网络分析结果揭示“U87和U251细胞差异基因”互作网络中包含1 467个节点和8 080对相互作用; “ART干预U87细胞效应基因”互作网络中包含1 352个节点和6 250对相互作用; “ART干预U251细胞效应基因”互作网络中包含160个节点和251对相互作用。接着, 网络拓扑特征值计算筛选数值均大于相应中位数的节点为关键网络靶标, 共得到454个ART敏感性和非敏感性细胞株差异基因的关键网络靶点(连接度: 6.00; 介度: 1 349.40; 紧密度: 386.80; 最大集团中心度: 8.00); 452个ART干预U87细胞的关键网络靶点(连接度: 6.00; 介度: 1 570.90; 紧密度: 351.50; 最大集团中心度: 7.00) 和40个ART干预U251细胞关键网络靶点(连接度: 6.00; 介度: 23.00; 紧密度: 30.80; 最大集团中心度: 4.00)。

生物功能挖掘结果表明, ART敏感性和非敏感性细胞株差异基因的关键网络靶点显著富集于免疫-炎症反应和细胞增殖分化等相关通路; ART干预U87细胞的关键网络靶点显著富集于细胞生长与死亡和DNA损伤修复等相关通路; ART干预U251细胞的关键网络靶点显著富集于与炎症和代谢相关的病理环节。其中, MAPK信号通路为3个对比组关键网络靶点共同富集的通路, 而凋亡为U87 vs U251和U87+ART vs U87对比组关键网络靶点共同富集的通路。本研究选取上述两条特殊通路做进一步分析, 发现其中的ATF4和DDIT3基因在给药ART前后的U87细胞中的表达分别由42.01和7.77上调为129.62和29.89 (ATF4:∣log₂FC = 1.65, P < 0.001; DDIT3:∣log₂FC∣= 1.96, P < 0.001); 而在给药ART前后的U251细胞中的表达分别由33.03和3.02上调为88.96和6.24 (ATF4:∣log₂FC∣ = 1.46, P < 0.001; DDIT3:∣log2FC∣ = 1.08, P < 0.001)。由此可见, ATF4和DDIT3基因在U87细胞中上调趋势更明显。两基因下游的PARP1在给药ART前后的U87细胞中表达由26.48下调为8.25 (∣log₂FC∣ = 1.65, P < 0.001), 而在U251中的表达无明显差异。3个基因均与细胞凋亡显著相关^[16-18], 因此本课题组推测, 与细胞凋亡有关的ATF4-DDIT3-PARP1信号轴可能是ART干预U87脑胶质瘤细胞的关键作用通路, 而PARP1由于在ART作用后的U87和U251中有不同的表达情况, 可能是U251脑胶质瘤细胞对药物产生耐药的重要靶标(图 3)。

4 ART与ATF4-DDIT3-PARP1信号轴中的相关蛋白均有较强的结合力

基于AutoDock v4.2.6和AutoDock Vina 1.1.2分子对接软件对ART与核心靶点之间的亲和力分析结果表明, ART与ATF4的对接分数是-4.57和-7.60 kcal·mol^-1; 与DDIT3的对接分数是-6.43和-6.50 kcal·mol^-1; 与PARP1的对接分数是-4.57和-6.90 kcal·mol^-1 (图 4)。接下来采用AutoDock Vina 1.1.2对上述3个靶标蛋白与其在PDB数据库收录的配体进行对接, 进一步验证ART与靶标蛋白的结合能力, 由于PDB数据库中未收录DDIT3的配体, 未进行对接验证。对接结果显示, ATF4与其配体PO4^3-和Mn²⁺的结合能分别为-3.90和-1.20 kcal·mol^-1; PARP1与其配体talazoparib的结合能为-14.30 kcal·mol^-1 (表 1)。结合上述对接结果可见, ART与该信号轴中的ATF4、PARP1和DDIT3具有较强的结合能力。

讨论

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在提高血脑屏障通过率的基础上更加科学全面地筛选合适的关键靶点或者相关通路, 对于颅内肿瘤药物的研发至关重要。本研究基于ART作用于两种人源脑胶质瘤细胞的体外细胞实验结果, 采用转录组测序获得差异表达基因, 并构建药效相关基因互作网络, 深入挖掘药物对U87和U251两种脑胶质瘤细胞敏感性差异的生物学内涵, 并进行分子对接验证青蒿琥酯与目标信号轴靶点的结合能力, 旨在为ART治疗脑胶质瘤提供有效的实验学依据。

基于两种人源脑胶质瘤细胞株(U87和U251) 的体外细胞活性和增殖实验表明, ART对U87细胞活性和增殖能力的抑制作用强于对U251细胞, 且ART可显著促进U87细胞凋亡, 而对U251效果不明显。为深入探索ART抗脑胶质瘤的药效和耐药分子内涵, 本研究对ART作用24 h前后U87和U251脑胶质瘤细胞进行转录组测序和网络药理学分析, 拓扑特征值计算和功能富集后发现, U87 vs U251、U87+ART vs U87和U251+ART vs U251组内差异基因均显著富集于多条与胶质瘤病理环节相关的信号通路; ART敏感性(U87细胞) 和非敏感性(U251细胞) 细胞株差异基因、ART干预U87细胞和ART干预U251细胞的关键网络靶标均显著富集于细胞凋亡相关的通路, 其中, ART干预脑胶质瘤细胞的效应靶标参与了ATF4-DDIT3-PARP1信号轴, 且PARP1在ART干预后U87细胞中的基因表达量显著低于给药前, 但在U251细胞给药前后无明显表达。ATF4和DDIT3在ART干预后的U87和U251中均表达上调, 而在U87细胞中上调趋势更为明显。分子对接分析得出ART与靶标通路中ATF4蛋白具有较强的亲和力。当ART与ATF4和DDIT3结合时, 线粒体凋亡途径被激活, 切割PARP1使其表达量降低, 抑制肿瘤细胞DNA损伤修复, 最终导致肿瘤细胞凋亡, 这与转录组测序和差异基因表达分析结果相符。

研究发现, ART可通过激活ATF4-CHOP (DDIT3) 信号轴, 发挥干预淋巴瘤的作用^[19], 表明ATF4可能为ART干预脑胶质瘤细胞活性的初始靶点。在脑胶质瘤中, 3种内质网跨膜蛋白所对应的3个分支, 蛋白激酶R样内质网激酶[protein kinase R (PKR)-like endoplasmic reticulum kinase, PERK] 通路、激活转录因子6 (activating transcription factor 6, ATF6) 通路、肌醇需要蛋白1 (inositol-requiring protein 1, IRE1) 通路均被持续性地高水平激活^[20], 极端的内质网应激可导致细胞死亡^[21]。ATF4作为正常细胞代谢和氧化还原过程的调节剂, 以及综合应激反应期间的主转录因子^[22], 与DDIT3协同促进与末端未展开蛋白质反应有关的基因表达来介导程序性细胞死亡, 并诱导细胞周期停滞和细胞凋亡以响应雌激素受体应激^[21]。其后, 作为内部凋亡刺激因子, 线粒体凋亡途径被激活。DDIT3通过抑制线粒体膜上Bcl-2家族的抗凋亡蛋白来调节线粒体外膜通透性。Bcl-2等插入线粒体膜中, 形成离子通道, 引起细胞色素C被释放到细胞质。进入胞质中的细胞色素C与Apaf-1结合, 通过招募激活pro-caspase-9, 形成caspase-9全酶^[23]。而caspase-9全酶可进一步激活caspase-3和caspase-7, 切割细胞中的PARP1, 最终诱导细胞凋亡^{[24, 25]}。这与本研究的转录组测序结果相符合, 即给药后表达上调的ATF4和DDIT3基因诱发了脑胶质瘤细胞的持续内质网应激, 进而引起细胞凋亡。PARP1仅在U87+ART vs U87中表达下调而在U251+ART vs U251中无显著变化, 提示其可能是U251脑胶质瘤细胞耐药的关键靶标。U87和U251两种细胞主要体现在TP53基因表型上的差异, U87细胞为野生型p53 (wtp53), 而U251为突变型p53 (mutp53)。wtp53具有肿瘤抑制功能, 而mutp53不仅无肿瘤抑制功能, 反而通过调控剩余wtp53促进肿瘤发生发展, 使得肿瘤细胞产生耐药^{[26, 27]}。mutp53和PARP1的基因表达水平呈正相关, 高表达可促进细胞增殖^[28]。此外, PARP1抑制剂与TMZ能共同抑制对TMZ耐药的脑胶质瘤生长, 可作为克服药物耐药性的潜在辅助剂^[29]。鉴此, 可推测U251细胞由于存在TP53突变, 使得PARP1降解被抑制, 肿瘤细胞损伤的DNA被修复从而产生耐药(图 5)。

综上所述, 本研究通过干湿实验相结合的研究策略, 初步揭示了ART干预U87脑胶质瘤细胞的潜能与其抑制肿瘤细胞内异常活化的ATF4-DDIT3-PARP1信号轴, 进而诱导细胞凋亡和抑制其增殖有关。相关研究结果将为临床对化疗药物产生耐药的脑胶质瘤患者提供新的潜在候选药物, 也为ART抗脑胶质瘤的临床应用提供科学依据。

作者贡献: 李涛负责数据分析、文献调研、文章撰写; 毛霞负责实验设计、数据分析、稿件修改; 张彦琼、林娜、黄丰负责实验指导、稿件修改; SATO Takashi、MIZUNO Koji、OKUYAMA Katsuki负责实验设计。

利益冲突: 本文作者均声明无利益冲突。

基金

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国家自然科学基金资助项目(8210143224)
中国中医科学院优秀青年科技人才(创新类)(ZZ15-YQ-029)

参考文献

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2023年第58卷第6期

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doi: 10.16438/j.0513-4870.2022-0821

接收时间：2022-07-05
首发时间：2025-11-21
出版时间：2023-06-12

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收稿日期：2022-07-05
修回日期：2022-11-23

基金

国家自然科学基金资助项目(8210143224)

中国中医科学院优秀青年科技人才(创新类)(ZZ15-YQ-029)

作者信息

1.云南中医药大学中药学院, 云南昆明 650500

2.中国中医科学院中药研究所, 北京 100700

3.东京药科大学生物化学系, 东京都八王子市 192-0392

通讯作者:

*毛霞, Tel: 86-10-64014411-2869, E-mail: xmao@icmm.ac.cn;

黄丰, Tel: 86-871-67429415, E-mail: 14030@ynutcm.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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