木犀草素对白血病K562/ADR细胞多药耐药的逆转作用及机制研究

doi:10.11958/20230722

天津医药 ›› 2023, Vol. 51 ›› Issue (12): 1321-1325.doi: 10.11958/20230722

木犀草素对白血病K562/ADR细胞多药耐药的逆转作用及机制研究

周欣宇¹(), 李京敏², 张婷¹, 贾秀红¹^,^△()

1.滨州医学院附属医院儿科（邮编256603）
2.滨州医学院基础医学院人体解剖学教研室

收稿日期:2023-05-18 修回日期:2023-07-20 出版日期:2023-12-15 发布日期:2023-12-22
通讯作者: ^△ E-mail：jiaxiuhong001@163.com
作者简介:周欣宇（1995），女，硕士在读，主要从事白血病的发病机制及治疗方面研究。E-mail：zhouxinyu002x@163.com
基金资助:
山东省自然科学基金资助项目(ZR2014HL032);山东省医药卫生科技发展计划项目(2017WS038)

Study on the mechanism of luteolin reversing multidrug resistance in leukemia K562/ADR cells

ZHOU Xinyu¹(), LI Jingmin², ZHANG Ting¹, JIA Xiuhong¹^,^△()

1. Department of Pediatrics, Binzhou Medical University Hospital, Binzhou 256603, China
2. Department of Human Anatomy, Basic Medical College, Binzhou Medical University

Received:2023-05-18 Revised:2023-07-20 Published:2023-12-15 Online:2023-12-22
Contact: ^△ E-mail：jiaxiuhong001@163.com

周欣宇, 李京敏, 张婷, 贾秀红. 木犀草素对白血病K562/ADR细胞多药耐药的逆转作用及机制研究[J]. 天津医药, 2023, 51(12): 1321-1325.

ZHOU Xinyu, LI Jingmin, ZHANG Ting, JIA Xiuhong. Study on the mechanism of luteolin reversing multidrug resistance in leukemia K562/ADR cells[J]. Tianjin Medical Journal, 2023, 51(12): 1321-1325.

摘要/Abstract

摘要：

目的探究木犀草素（Lut）对慢性髓系白血病K562/ADR细胞多药耐药性的逆转作用及其机制。方法 K562和K562/ADR细胞经不同浓度阿霉素（ADR）处理24 h后，采用CCK-8实验检测K562/ADR细胞耐药倍数。Lut单独或联合ADR作用K562/ADR细胞24 h后，采用CCK-8实验检测Lut的细胞毒性及对ADR的增敏作用。取对数生长期K562/ADR细胞分为0 μmol/L Lut组、2 μmol/L Lut组、4 μmol/L Lut组，采用流式细胞术检测细胞内ADR蓄积量的变化；RT-PCR法和Western blot法分别检测核因子E2相关因子2（Nrf2）、多药耐药相关蛋白1（MRP1）、P-糖蛋白（P-gp）、谷胱甘肽-S-转移酶pi（GST-pi）mRNA和蛋白的表达；谷胱甘肽（GSH）试剂盒检测细胞内GSH的含量。结果与K562细胞相比，K562/ADR细胞株对ADR具有明显的耐药性，耐药倍数为53.69倍。与0 μmol/L Lut相比，不同浓度Lut作用于K562/ADR细胞后，细胞生长均受到不同程度的抑制（P<0.05），其中2、4 μmol/L Lut对K562/ADR细胞的增殖抑制率<10%，为无毒性的Lut浓度。与0 μmol/L Lut组相比，2、4 μmol/L Lut组可明显增强ADR对K562/ADR的细胞增殖抑制率，增加细胞内ADR蓄积量，提高逆转耐药倍数，降低细胞内GSH含量，下调细胞中MRP1、P-gp、GST-pi、Nrf2 mRNA及蛋白的表达（P<0.05）；且4 μmol/L Lut作用效果较2 μmol/L Lut更显著。结论 Lut可抑制K562/ADR细胞增殖，逆转其对ADR的耐药性，其机制可能与Lut下调Nrf2、MRP1、P-gp、GST-pi表达，进而增加细胞内ADR蓄积量有关。

关键词: 白血病, 髓系, 慢性, BCR-ABL阳性, 木犀草素, 抗药性, 多药, 核因子E2相关因子2, 多药耐药相关蛋白1, P-糖蛋白, 谷胱甘肽-S-转移酶pi

Abstract:

Objective To investigate the mechanism of luteolin′s (Lut) reversal effect on multidrug resistance of chronic myeloid leukemia K562/ADR cells. Methods CCK-8 assay was used to detect drug resistance in K562 and K562/ADR cells 24 hours after treatment with different doses of adriamycin (ADR). CCK-8 assay was used to assess the cytotoxicity and sensitizing effect of Lut on ADR after K562/ADR cells were treated with Lut alone or in combination with ADR for 24 hours. K562/ADR cells in logarithmic growth phase were separated into three group: 0 μmol/L Lut, 2 μmol/L and 4 μmol/L Lut groups. ADR accumulation in cells was measured using flow cytometry. Nuclear factor erythroid-2-related factor 2 (Nrf2), multidrug resistance associated protein 1 (MRP1), P-glycoprotein (P-gp) and glutathione-S-transferase-PI (GST-pi) mRNA and protein expressions were identified using RT-PCR and Western blot assay. Glutathione (GSH) kit was used to detect intracellular GSH content. Results Compared with K562 cells, K562/ADR cell line was significantly resistant to ADR, and the drug resistance was 53.69 times. K562/ADR cell proliferation was decreased to variable degrees by different doses of Lut when compared to the 0 μmol/L Lut group (P<0.05). The proliferation inhibition rates of K562/ADR cells treated with 2 and 4 μmol/L Lut were less than 10%, indicating that the concentration of Lut was non-toxic. Compared with the 0 μmol/L Lut group, the 2 μmol/L Lut group and the 4 μmol/L Lut group showed significantly increased ADR growth inhibition rate on K562/ADR and increased accumulation of ADR in cells, improved the reversal resistance fold, and decreased GSH content in cells. MRP1, P-gp, GST-pi and Nrf2 mRNA and protein expression were reduced in cells (P<0.05). The effect of 4 mol/L Lut was greater than that of 2 mol/L Lut. Conclusion Lut may decrease K562/ADR cell proliferation and reverse ADR medication resistance. The mechanism could be connected to the downregulation of Nrf2, MRP1, P-gp and GST-pi expression, which leads to an increase in ADR accumulation in K562/ADR cells.

Key words: leukemia, myelogenous, chronic, BCR-ABL positive, Luteolin, drug resistance, multiple, nuclear factor erythroid2-related factor 2, multidrug resistance associated protein 1, P-glycoprotein, glutathione-S-transferase-PI

中图分类号:

R733.72

图/表 5

表1 RT-PCR引物序列

Tab.1 Primer sequence of RT-PCR

基因名称	引物序列（5'→3'）	产物大小/bp
MRP1	上游：CCTGGGCATTTCACAAGGGAT	139
MRP1	下游：GGTCCGCTCAAAGAAGCTCA	139
P-gp	上游：CCACAGAGGGGATGGTCAGT	142
P-gp	下游：GGTCCGCTCAAAGAAGCTCA	142
GST-pi	上游：AGCCTTTTGAGACCCTGCTGC	122
GST-pi	下游：AGGGGCTAGGACCTCATGGAT	122
Nrf2	上游：TAGTGCCCCTGGAAGTGTCAA	112
Nrf2	下游：ATCATGCACGTGAGTGCTCTG	112
GAPDH	上游：GCACCGTCAAGGCTGAGAAC	138
GAPDH	下游：TGGTGAAGACGCCAGTGGA	138

图1 不同浓度Lut对K562/ADR细胞ADR敏感性的影响 n=3，ADR质量浓度为16、32、64、128 mg/L的F值分别为9.425、14.110、37.480和14.140，均P<0.05；a与0 μmol/L+ADR组比较，b与2 μmol/L+ADR组比较，P<0.05。

Fig.1 Effect of different concentrations of Lut on the sensitivity of K562/ADR cells to ADR

表2 各组MRRP1、P-gp、Nrf2、GST-pi mRNA相对表达水平比较

Tab.2 Comparison of relative expression levels of MRRP1, P-gp, Nrf2 and GST-pi mRNA between the three groups （n=3，$\bar{x}±s$）

组别	MRP1	P-gp	Nrf2	GST-pi
0 μmol/L Lut组	1	1	1	1
2 μmol/L Lut组	0.76±0.05^a	0.53±0.07^a	0.57±0.14^a	0.88±0.04^a
4 μmol/L Lut组	0.67±0.02^ab	0.36±0.05^ab	0.35±0.20^a	0.64±0.08^ab
F	101.700^＊	130.300^＊	15.650^＊	48.690^＊

图2 Western blot检测各组细胞MRP1、P-gp、Nrf2、GST-pi蛋白表达 A、B、C分别为1、2、4 μmol/L Lut组。

Fig.2 The expressions of MRP1, P-gp, Nrf2 and GST-pi detected by Western blot assay

表3 各组细胞MRP1、P-gp、Nrf2、GST-pi蛋白相对表达水平比较

Tab.3 Comparison of relative expression levels of MRP1, P-gp, Nrf2 and GST-pi proteins between the three groups （n=3，$\bar{x}±s$）

组别	MRP1	P-gp	Nrf2	GST-pi
0 μmol/L Lut组	1.15±0.10	1.42±0.03	1.89±0.19^a	1.20±0.11
2 μmol/L Lut组	0.78±0.13^a	1.04±0.03^a	1.18±0.17^a	0.78±0.05^a
4 μmol/L Lut组	0.51±0.06^ab	0.69±0.06^ab	0.85±0.17^a	0.47±0.07^ab
F	31.370^＊	237.800^＊	27.090^＊	63.940^＊

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木犀草素对白血病K562/ADR细胞多药耐药的逆转作用及机制研究

Study on the mechanism of luteolin reversing multidrug resistance in leukemia K562/ADR cells

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