芪贞归脾汤对淋巴瘤细胞株生物学行为的影响及其机制探讨
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摘要
目的 :探讨芪贞归脾汤对淋巴瘤细胞增殖、凋亡、细胞周期、迁移和侵袭的影响,并通过高通量组学技术探索其可能的分子作用机制。方法:采用不同浓度的芪贞归脾汤作用于淋巴瘤Jurkat、SU-DHL-4和Raji细胞株;CCK-8法检测细胞的增殖抑制率;FCM法检测细胞凋亡率及细胞周期;Transwell小室法检测细胞的迁移和侵袭能力;采用PrimeView人类基因表达谱芯片(PrimeView Human Gene Expression Array)检测基因表达谱变化;应用IngenuityPathwayAnalysis(IPA)进行经典通路分析;选择有差异表达的6个基因[磷脂酰肌醇3-激酶催化亚单位α(phosphatidylinositol3-kinasecatalyticsubunitalpha,PIK 3CA)、B细胞κ轻肽基因增强子核因子1(nuclearfactorofkappalightpolypeptide geneenhancerinB-cells1,NFKB 1)、B细胞κ轻肽基因增强子抑制因子,激酶β(inhibitor of kappa light polypeptide gene enhancer in B-cells,kinase beta,IKBKB)、B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)、Janus激酶2(Januskinase2,JAK 2)和信号转导及转录激活因子3(signaltransducer and activator of transcription 3,STAT 3)]进行实时荧光定量PCR法验证。结果 :质量浓度为5～25 mg/mL的芪贞归脾汤对Jurkat、SU-DHL-4和Raji细胞株均有明显的增殖抑制作用(P值均<0.01)。芪贞归脾汤质量浓度为20和25 mg/mL组Jurkat及Raji细胞的凋亡率显著升高(P值均<0.01)。10、15和20mg/mL芪贞归脾汤作用48h后,3株淋巴瘤细胞的细胞周期均被阻滞在G2/M期(P值均<0.01)。15mg/mL芪贞归脾汤可显著抑制Jurkat及Raji细胞的迁移和侵袭能力(P值均<0.05)。Jurkat细胞在15mg/mL芪贞归脾汤作用48h后,共有648个基因表达上调和1022个基因表达下调。基于IPA的经典通路分析结果显示,差异基因显著富集的信号通路中磷脂酰肌醇3-激酶(phosphoinositide3-kinase,PI3K)-蛋白激酶B(protein kinaseB,PKB,又称Akt)信号通路(P <0.001)及JAK/STAT信号通路(P <0.05)明显受到抑制。PIK3CA、NFKB1、IKBKB、Bcl-2、JAK2和STAT3 mRNA的表达水平均明显下调(STAT3 mRNA为P <0.05,其余各基因的P值均<0.01),与芯片结果一致。结论 :中药芪贞归脾汤可以调控淋巴瘤细胞的生物学行为,并通过多基因及多通路发挥抗淋巴瘤的作用。
Objective: To investigate the effects of Qi-Zhen-Gui-Pi decoction on the proliferation, apoptosis, cell cycle, migration and invasion of lymphoma cells, and to explore the possible molecular mechanism using high throughput omics technology. Methods: The human lymphoma cell lines(Jurkat, SU-DHL-4, and Raji) were treated with different concentrations of Qi-Zhen-Gui-Pi decoction. The proliferation inhibitory rate of lymphoma cells was detected by CCK-8 method. The changes of apoptosis and cell cycle distribution were detected by FCM assay. The migration and invasion abilities of lymphoma cells were detected by Transwell chamber assay. The gene expression profiles were detected by PrimeView Human Gene Expression Array. Ingenuity Pathway Analysis(IPA) was used to analyze the classical signaling pathway. The expression levels of differentially expressed genes including phosphatidylinositol 3-kinase catalytic subunit alpha(PIK 3 CA), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1(NFKB 1), inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta(IKBKB), B-cell lymphoma-2(Bcl-2), Janus kinase 2(JAK2) and signal transducer and activator of transcription 3(STAT3) were detected by real-time fluorescent quantitative PCR.Results: After the treatment with different concentrations of Qi-Zhen-Gui-Pi decoction(5-25 mg/mL), the proliferation of Jurkat, SU-DHL-4 and Raji cells was significantly inhibited(all P < 0.01). After the treatment with 20 mg/mL and 25 mg/mL Qi-Zhen-Gui-Pi decoction, the apoptosis rates of Jurkat and Raji cells were significantly increased(all P < 0.01). After the treatment with different concentrations of Qi-Zhen-Gui-Pi decoction(10, 15 and 20 mg/mL) for 48 h, the cell cycle of Jurkat, SU-DHL-4 and Raji cells was arrested in G2/M phase(all P < 0.01). The migration and invasion activities of Jurkat and Raji cells were significantly suppressed by Qi-Zhen-Gui-Pi decoction(15 mg/mL)(all P < 0.01). According to the gene expression profiling data, 648 genes were up-regulated, and 1 022 genes were downregulated after Jurkat cells were treated with Qi-Zhen-Gui-Pi decoction for 48 h. Based on IPA pathway analysis, the phosphoinositide3-kinase(PI3 K)/protein kinase B(PKB, Akt) signaling pathway and JAK/STAT signaling pathway were significantly blocked among the enriched pathways of differentially expressed genes(P < 0.001, P < 0.05). The expression levels of PIK3 CA, NFKB1, IKBKB, Bcl-2, JAK2 and STAT3 mRNAs were significantly down-regulated(PSTAT3 < 0.05, Pothers < 0.01).Conclusion: Qi-Zhen-Gui-Pi decoction can regulate the biological behavior of lymphoma cell lines, and play an anti-lymphoma role through multiple genes and signaling pathways.
Objective: To investigate the effects of Qi-Zhen-Gui-Pi decoction on the proliferation, apoptosis, cell cycle, migration and invasion of lymphoma cells, and to explore the possible molecular mechanism using high throughput omics technology. Methods: The human lymphoma cell lines(Jurkat, SU-DHL-4, and Raji) were treated with different concentrations of Qi-Zhen-Gui-Pi decoction. The proliferation inhibitory rate of lymphoma cells was detected by CCK-8 method. The changes of apoptosis and cell cycle distribution were detected by FCM assay. The migration and invasion abilities of lymphoma cells were detected by Transwell chamber assay. The gene expression profiles were detected by PrimeView Human Gene Expression Array. Ingenuity Pathway Analysis(IPA) was used to analyze the classical signaling pathway. The expression levels of differentially expressed genes including phosphatidylinositol 3-kinase catalytic subunit alpha(PIK 3 CA), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1(NFKB 1), inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta(IKBKB), B-cell lymphoma-2(Bcl-2), Janus kinase 2(JAK2) and signal transducer and activator of transcription 3(STAT3) were detected by real-time fluorescent quantitative PCR.Results: After the treatment with different concentrations of Qi-Zhen-Gui-Pi decoction(5-25 mg/mL), the proliferation of Jurkat, SU-DHL-4 and Raji cells was significantly inhibited(all P < 0.01). After the treatment with 20 mg/mL and 25 mg/mL Qi-Zhen-Gui-Pi decoction, the apoptosis rates of Jurkat and Raji cells were significantly increased(all P < 0.01). After the treatment with different concentrations of Qi-Zhen-Gui-Pi decoction(10, 15 and 20 mg/mL) for 48 h, the cell cycle of Jurkat, SU-DHL-4 and Raji cells was arrested in G2/M phase(all P < 0.01). The migration and invasion activities of Jurkat and Raji cells were significantly suppressed by Qi-Zhen-Gui-Pi decoction(15 mg/mL)(all P < 0.01). According to the gene expression profiling data, 648 genes were up-regulated, and 1 022 genes were downregulated after Jurkat cells were treated with Qi-Zhen-Gui-Pi decoction for 48 h. Based on IPA pathway analysis, the phosphoinositide3-kinase(PI3 K)/protein kinase B(PKB, Akt) signaling pathway and JAK/STAT signaling pathway were significantly blocked among the enriched pathways of differentially expressed genes(P < 0.001, P < 0.05). The expression levels of PIK3 CA, NFKB1, IKBKB, Bcl-2, JAK2 and STAT3 mRNAs were significantly down-regulated(PSTAT3 < 0.05, Pothers < 0.01).Conclusion: Qi-Zhen-Gui-Pi decoction can regulate the biological behavior of lymphoma cell lines, and play an anti-lymphoma role through multiple genes and signaling pathways.
引文
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[23] SONG TL, NAIRISMAGI ML, LAURENSIA Y,et al. Oncogenic activation of the STAT3pathway drives PD-L1 expression in natural killer/T-cell lymphoma[J].Blood, 2018, 132(1 1):1146-11 58.
[24] TIACCI E, LADEWIG E, SCHIAVONI G,et al. Pervasive mutations of JAKSTAT pathway genes in classical Hodgkin lymphoma[J]. Blood, 2018,131(22):2454-2465.
[2] SWERDLOW SH, CAMPO E, HARRIS NL,et al. World Health Organization Classification of tumors of hematopoietic and lymphoid tissues[M]. Revised 4th ed. Lyon,France:International Agency for Reseach on Cancer, 201 7.
[3] XI SY, MINUK GY. Role of traditional Chinese medicine in the management of patients with hepatocellular carcinoma[J]. World J Hepatol, 2018,10(11):799-806.
[4] MENG Q, PAN J,LIU Y,et al. Antitumour effects of polysaccharide extracted from acanthopanax se nt i cos u s and cell-mediated immunity[J]. Exp Ther Med, 2018,15(2):1 694-1 701.
[5] HOU C, ZHOU DH, WU YJ, et al. In vitro and in vivo inhibitory effect of Gujin Xiaoliu Tang in non-small cell lung cancer[J]. Evid Based Complement Alternat Med, 201 8, 201 8:8936108.
[6]吴娇,王聪.黄芪的化学成分及药理作用研究进展[J].新乡医学院学报,2018,35(9):755-760.
[7] GAO L, LI C, WANG Z, et al. Ligustri lucidi fructus as a traditional Chinese medicine:a review of its phytochemistry and pharmacology[J].Nat Prod Res, 2015, 29(6):493-510.
[8] WANG ZY, NIXON DW. Licorice and cancer[J]. Nutr Cancer, 2001,39(1):1-11.
[9] XU J, SONG Z, GUO Q, et al. Synergistic effect and molecular mechanisms of traditional Chinoese medicine onregulating tumor microenvironment and cancer cells[J]. Biomed Res Int,2016, 2016:1490738.
[10] GUO L, BAI SP, ZHAO L, et al.Astragalus polysaccharide injection integrated with vinorelbine and cisplatin for patients with advanced non-small cell lung cancer:effects on quality of life and survival[J]. Med Oncol, 2012, 29(3):1656-1662.
[11] WU Y, WANG D, YANG X, et al.Traditional Chinese medicine Gegen Qinlian decoction ameliorates irinotecan chemotherapy-induced gut toxicity in mice[J]. Biomed Pharmacother,2019, 109:2252-2261.
[12] KIM C, LEE SG, YANG WM, et al.Formononetin-induced oxidative stress abrogates the activation of STAT3/5 signaling axis and suppresses the tumor growth in multiple myeloma preclinical model[J].Cancer Lett, 2018, 431:123-141.
[13] ZHANG D, ZHUANG Y, PAN J, et al.Investigation of effects and mechanisms of total flavonoids of astragalus and calycosin on human erythroleukemia cells[J]. Oxid Med Cell Longev, 201 2, 201 2:209843.
[14]李杰,许良中,朱伟萍,等.熊果酸与齐墩果酸体外抗Jurkat淋巴瘤细胞的研究[J].中国癌症杂志,1999, 9(5):395-397.
[15] HE SQ, GAO M, FU YF, et al.Glycyrrhizic acid inhibits leukemia cell growth and migration via blocking AKT/mTOR/STAT3 signaling[J].Int J Clin Exp Pathol, 2015,8(5):5175-5181.
[16] PIRZADEH S, FAKHARI S,JALILI A, et al.Glycyrrhetinic acid induces apoptosis in leukemic HL60 cells through upregulating of CD95/CD1 78[J]. Int J Mol Cell Med, 2014, 3(4):272-278.
[17] PANG Z, ZHI-YAN Z, WANG W, et al.The advances in research on the pharmacological effects of Fructus Ligustri Lucidi[J]. Biomed Res Int,201 5, 201 5:281873.
[18] YANG R, WANG LQ, YUAN BC, et al. The pharmacological activities of licoriceU].Planta Med, 2015, 81(1 8):1654-1 669.
[19]曲琦,黄洪晖,陈芳源,等.经典Wnt信号通路在非霍奇金淋巴瘤细胞株中作用的初步研究[J].肿瘤,2013,33(2):144-149.
[20] CUI W, CAI Y, WANG W, et al. Frequent copy number variations of PI3K/AKT pathway and aberrant protein expressions of PI3K subunits are associated with inferior survival in diffuse large B cell lymphoma[J].J Transl Med, 2014, 12:10.
[21] CAI Q, DENG H, XIE D, et al.Phosphorylated AKT protein is overexpressed in human peripheral T-cell lymphomas and predicts decreased patient survival[J]. Clin Lymphoma Myeloma Leuk,2012,12(2):106-112.
[22] LAMPSON BL, BROWN JR. PI3Kδ-selective and PI3Ka/δ-combinatorial inhibitors in clinical development for B-cell non-hodgkin lymphoma[J].Expert Op in Investig Drugs, 201 7,26(11):1267-1279.
[23] SONG TL, NAIRISMAGI ML, LAURENSIA Y,et al. Oncogenic activation of the STAT3pathway drives PD-L1 expression in natural killer/T-cell lymphoma[J].Blood, 2018, 132(1 1):1146-11 58.
[24] TIACCI E, LADEWIG E, SCHIAVONI G,et al. Pervasive mutations of JAKSTAT pathway genes in classical Hodgkin lymphoma[J]. Blood, 2018,131(22):2454-2465.