氢水通过上调核因子红细胞2相关因子2/血红素加氧酶1表达抑制顺铂致肝细胞损伤作用研究

doi:10.12114/j.issn.1007-9572.2024.0093

中国全科医学 ›› 2026, Vol. 29 ›› Issue (29): 4288-4294.DOI: 10.12114/j.issn.1007-9572.2024.0093

氢水通过上调核因子红细胞2相关因子2/血红素加氧酶1表达抑制顺铂致肝细胞损伤作用研究

王晗¹, 陈剑华², 宋时莉³, 郝珍³, 霍忠超⁴, 李沙², 李卫霞²^,^*()

1．056038　河北省邯郸市，河北工程大学临床医学院
2．056038　河北省邯郸市，河北工程大学医学院
3．056027　河北省邯郸市，中国船舶集团有限公司第七一八研究所制氢设备工程部
4．056038　河北省邯郸市，河北工程大学附属医院肿瘤中心

收稿日期:2024-04-10 修回日期:2024-07-10 出版日期:2026-10-15 发布日期:2026-09-02
通讯作者: 李卫霞
作者贡献：
王晗负责课题实施与论文的撰写；陈剑华负责收集实验数据与整理并进行统计学分析；宋时莉、郝珍负责氢水制备及浓度检测；霍忠超、李沙负责论文审校与修订；李卫霞负责文章的构思与设计，并对文章负责。
基金资助:
河北省重点研发计划项目(223777128D); 邯郸市科学技术研究与发展计划项目(23422083045)

Inhibition Effect of Hydrogen Water on Cisplatin Induced Hepatocyte Injury via Upregulating NFE2L2/HMOX1 Expression

WANG Han¹, CHEN Jianhua², SONG Shili³, HAO Zhen³, HUO Zhongchao⁴, LI Sha², LI Weixia²^,^*()

1. Clinical Medicine College, Hebei University of Engineering, Handan 056038, China
2. Medicine College, Hebei University of Engineering, Handan 056038, China
3. Hydrogen Production Equipment Engineering Department, 718th Research Institute of CSIC, Handan 056027, China
4. Cancer Center, Affiliated Hospital of Hebei University of Engineering, Handan 056038, China

Received:2024-04-10 Revised:2024-07-10 Published:2026-10-15 Online:2026-09-02
Contact: LI Weixia

摘要/Abstract

摘要： 背景顺铂诱导的药物性肝损伤是肿瘤化疗常见的毒副作用，严重影响了患者临床治疗。氢作为一种选择性抗氧化剂，在多种损伤相关的疾病中均具有良好的辅助治疗作用，但其在顺铂诱导的肝损伤中的预防作用及其机制尚未明确。目的探讨氢水对顺铂致肝细胞氧化应激性损伤的保护作用及作用机制。方法于2022年6月—2023年6月取人正常肝细胞株WRL68分为对照组、顺铂组、氢水组、氢水+顺铂组。对照组采用含10%胎牛血清的RPMI-1640培养基培养48 h；顺铂组采用含10%胎牛血清的RPMI-1640培养基培养24 h后，加入3 mg/L顺铂继续培养24 h；氢水组采用0.6 mg/L氢水培养基培养48 h；氢水+顺铂组采用0.6 mg/L氢水培养基培养24 h后，加入3 mg/L顺铂继续培养24 h。收集各组细胞，采用CCK-8法检测细胞活力，流式细胞仪检测细胞凋亡率，罗丹明123荧光探针染色法检测线粒体膜电位（MMP），DCFH-DA荧光探针染色法检测细胞内活性氧（ROS），Western blotting和RT-qPCR检测细胞内核因子红细胞2相关因子2（NFE2L2）、血红素加氧酶1（HMOX1）蛋白及mRNA表达水平。结果与对照组相比，顺铂组WRL68细胞凋亡率、ROS水平升高（P<0.05），MMP水平及NFE2L2、HMOX1的蛋白及mRNA水平降低（P<0.05）；氢水组WRL68细胞凋亡率、ROS水平与对照组相比，差异均无统计学意义（P>0.05）；氢水组MMP水平及NFE2L2、HMOX1的蛋白及mRNA水平均高于对照组（P<0.05）。与顺铂组相比，氢水+顺铂组WRL68细胞凋亡率、ROS水平均降低（P<0.05），MMP水平及NFE2L2、HMOX1的蛋白及mRNA表达水平均升高（P<0.05）。结论氢水可通过激活NFE2L2/HMOX1信号通路，提高MMP，抑制顺铂引起的肝细胞氧化应激性损伤，从而减轻药物性肝损伤。

关键词: 药物性肝损伤, 顺铂, 氢水, 氧化应激, NFE2L2/HMOX1信号通路

Abstract:

Background

Cisplatin induced drug-induced liver injury is a common toxic side effects in tumor chemotherapy, which seriously disturbs the clinical treatment of patients. Hydrogen, as a selective antioxidant, has good adjunctive therapeutic effects in various injury related diseases, but its preventive effect and mechanism in cisplatin induced liver injury are not clear.

Objective

To study the protective effect and mechanism of hydrogen water on oxidative stress injury of hepatocytes induced by cisplatin.

Methods

From June 2022 to June 2023, human normal liver cell line WRL68 were divided into control group, cisplatin group, hydrogen water group, and hydrogen water plus cisplatin group. Control group was cultured with RPMI-1640 medium containing 10% fetal bovine serum for 48 h. Cisplatin group was cultured with RPMI-1640 medium containing 10% fetal bovine serum for 24 h, then added 3 mg/L cisplatin and continued culturing for 24 h. Hydrogen water group was cultured with 0.6 mg/L hydrogen water medium for 48 h. Hydrogen water plus cisplatin group was cultured with 0.6 mg/L hydrogen water medium for 24 h, then added 3 mg/L cisplatin and continued culturing for 24 h. Then collect cells of each gorup, and hepatocyte activity was detected with CCK-8 method. Cell apoptosis was detected by flow cytometry. Mitochondrial membrane potential (MMP) was detected via Rhodamine 123 fluorescence staining method, and intracellular reactive oxygen species (ROS) levels were detected via DCFH-DA fluorescence probe staining method, the proteins and mRNA expression of nuclear factor erythroid 2-related factor 2 (NFE2L2) and heme oxygenase-1 (HMOX1) were detected by western blotting and RT-qPCR.

Results

Compared with the control group, the apoptosis rate and ROS level of cisplatin group increased (P<0.05), while the relative levels of MMP, NFE2L2, HMOX1 protein and mRNA decreased (P<0.05). There was no statistically significant difference in cell apoptosis rate and ROS level between hydrogen water group and control group (P>0.05), while the relative levels of MMP, NFE2L2, and HMOX1 proteins and mRNA increased (P<0.05). Compared with cisplatin group, hydrogen water plus cisplatin group decreased in cell apoptosis rate and ROS level (P<0.05), while the relative levels of MMP, NFE2L2, HMOX1 proteins and mRNA increased (P<0.05).

Conclusion

Hydrogen water can inhibit the oxidative stress injury in cisplatin induced hepatocytes via activating NFE2L2/HMOX1 signaling pathway, and improving MMP, then alleviate drug-induced liver injury.

Key words: Drug-induced liver injury, Cisplatin, Hydrogen water, Oxidative stress, NFE2L2/HMOX1 signaling pathway

中图分类号:

R 575.1 R 979.19

王晗,陈剑华,宋时莉,等. 氢水通过上调核因子红细胞2相关因子2/血红素加氧酶1表达抑制顺铂致肝细胞损伤作用研究[J]. 中国全科医学, 2026, 29(29): 4288-4294. DOI: 10.12114/j.issn.1007-9572.2024.0093.
WANG Han,CHEN Jianhua,SONG Shili, et al. Inhibition Effect of Hydrogen Water on Cisplatin Induced Hepatocyte Injury via Upregulating NFE2L2/HMOX1 Expression[J]. Chinese General Practice, 2026, 29(29): 4288-4294. DOI: 10.12114/j.issn.1007-9572.2024.0093.

图/表 9

表1 RT-qPCR引物序列

Table 1 RT-qPCR primer sequence

引物名称	基因编号	引物序列（5'-3'）	产物长度（bp）
NFE2L2	NM_001313903.2	上游：AGTCCAGAAGCCAAACTGACAGAAG	135
		下游：GGAGAGGATGCTGCTGAAGGAATC
HMOX1	NM_002133.3	上游：CTTCAAGCTGGTGATGGCCT	97
		下游：GTAGACAGGGGCGAAGACTG
ACTB	NM_001101.5	上游：GCACTCTTCCAGCCTTCCTTCC	93
		下游：GCGGATGTCCACGTCACACTTC

表2 不同浓度顺铂处理WRL68细胞活力比较（±s，%，n=3）

Table 2 Cell activity comparison of WRL68 treated by different concentrations of cisplatin

组别	细胞活力
对照组	100.00±0
顺铂1.25 mg/L亚组	88.55±1.48^a
顺铂2.50 mg/L亚组	68.59±2.68^a
顺铂5.00 mg/L亚组	38.95±2.15^a
顺铂10.00 mg/L亚组	23.27±1.52^a
顺铂20.00 mg/L亚组	10.65±1.98^a
F值	1 168.00
P值	<0.001

表3 氢水预处理后各组WRL68细胞活力比较（±s，%，n=3）

Table 3 Comparison of survival rates of WRL68 cells in different groups after hydrogen water pretreatment

组别	细胞活力
对照组	100.00±0.04
顺铂组	59.93±2.89^a
低浓度氢水组	100.90±1.19
低浓度氢水+顺铂组	77.50±3.67^ab
中浓度氢水组	99.04±1.68
中浓度氢水+顺铂组	72.28±5.94^ab
高浓度氢水组	103.10±1.53
高浓度氢水+顺铂组	74.15±4.43^ab
F值	80.590
P值	<0.001

表4 各组WRL68细胞凋亡率及MMP、ROS水平比较（±s，n=3）

Table 4 Comparison of apoptosis rate and level of MMP and ROS in each group of WRL68 cells

组别	细胞凋亡率（%）	MMP	ROS
对照组	2.49±0.38	1.00±0.05	1.00±0.15
顺铂组	32.90±1.97^a	0.35±0.01^a	6.52±0.99^a
氢水组	2.67±0.60^b	1.41±0.02^ab	1.05±0.18^b
氢水+顺铂组	23.15±1.19^ab	0.62±0.02^ab	4.00±0.40^ab
F值	479.000	702.600	70.360
P值	<0.001	<0.001	<0.001

图1 流式细胞仪检测WRL68细胞凋亡率注：A~D分别表示对照组、顺铂组、氢水组、氢水+顺铂组；PI=碘化丙啶，Annexin V-FITC=膜联蛋白V-异硫氰酸荧光素。

Figure 1 Apoptosis rate of WRL68 cells detected by flow cytometry

图2 荧光显微检测各组细胞MMP和ROS水平（×100）注：A为光镜下各组细胞状态，B为荧光显微检测各组细胞线粒体膜电位（MMP）水平，C为荧光显微检测各组细胞活性氧（ROS）水平。

Figure 2 The levels of MMP and ROS in hepatocytes detected by microfluorometry

表5 各组WRL68细胞NFE2L2与HMOX1 mRNA水平比较（±s，n=3）

Table 5 Comparison of relative levels of NFE2L2 and HMOX1 in each group of WRL68 cells

组别	蛋白		mRNA
组别	NFE2L2	HMOX1	NFE2L2	HMOX1
对照组	1.00±0.15	1.00±0.15	1.00±0.00	1.00±0.00
顺铂组	0.35±0.07^a	0.48±0.07^a	0.27±0.04^a	0.31±0.01^a
氢水组	2.54±0.09^ab	3.05±0.07^ab	1.56±0.33^ab	1.85±0.42^ab
氢水+顺铂组	0.89±0.14^b	2.19±0.11^b	0.90±0.19^b	1.29±0.27^b
F值	194.600	386.900	22.670	19.720
P值	<0.001	<0.001	<0.001	0.001

图3 Western blotting检测各组细胞NFE2L2与HMOX1的蛋白水平注：NFE2L2=核因子红细胞2相关因子2，ACTB=肌动蛋白β，HMOX1=血红素加氧酶1；A为Western blotting检测各组细胞NFE2L2蛋白水平，B为Western blotting检测各组细胞HMOX1蛋白水平。

Figure 3 The protein levels of NFE2L2 and HMOX1 in hepatocytes detected by western blotting

图4 氢水缓解顺铂诱导的肝细胞氧化应激性损伤机制示意图注：DDP=顺铂，OS=氧化应激，·O2-=超氧阴离子自由基，SOD=超氧化物歧化酶，H2O2=过氧化氢，NO=一氧化氮，ONOO-=过氧亚硝基阴离子，·OH=羟自由基，Keap1= Kelch样ECH相关蛋白1，sMaf=小肌肉筋膜纤维肉瘤，ARE=抗氧化反应元件。

Figure 4 Schematic diagram of hydrogen water alleviating cisplatin induced oxidative stress injury of hepatocytes

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氢水通过上调核因子红细胞2相关因子2/血红素加氧酶1表达抑制顺铂致肝细胞损伤作用研究

Inhibition Effect of Hydrogen Water on Cisplatin Induced Hepatocyte Injury via Upregulating NFE2L2/HMOX1 Expression

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