肝爽颗粒浸膏减轻对乙酰氨基酚所致肝细胞损伤的作用机制

吴桥; 余朋飞; 毕研贞; 王宝增; 王子璇; 李志杰; 陈煜; 段钟平

doi:10.3969/j.issn.1001-5256.2021.01.024

肝爽颗粒浸膏减轻对乙酰氨基酚所致肝细胞损伤的作用机制

DOI: 10.3969/j.issn.1001-5256.2021.01.024

吴桥^{1, 2},
余朋飞¹,
毕研贞¹,
王宝增²,
王子璇²,
李志杰³,
陈煜^1, ,,
段钟平^1, ,

1.
首都医科大学附属北京佑安医院 a.疑难肝病及人工肝中心, b.肝衰竭与人工肝治疗研究北京市重点实验室，北京100069
2.
首都医科大学附属北京天坛医院感染中心，北京 100070
3.
解放军总医院第五医学中心肝胆外科中心，北京 100039

基金项目:

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2012ZX10002004-006);

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2017ZX10203201-005);

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2017ZX10201201-001-001);

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2017ZX10201201-002-002);

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2017ZX10201201-004-002);

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2017ZX10202203-006-001);

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2017ZX10302201-004-002);

北京市医院管理局“登峰”人才培养计划基金项目 (DFL20151601);

北京市医院管理局临床医学发展专项 (ZYLX201806)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突，特此声明。

作者贡献声明：吴桥负责课题设计，资料分析，撰写论文; 余朋飞负责细胞实验、指标检测; 毕研贞负责高内涵检测及ROS数据的采集; 王宝增、王子璇、李志杰参与收集数据，修改论文; 陈煜、段钟平负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

作者简介:
吴桥(1989—)，女，医学博士，主要从事肝病及感染性疾病研究

余朋飞(1992—)，男，主要从事肝病研究

通信作者:
陈煜，chybeyond@163.com

段钟平，duan2517@163.com

二者对本文贡献相同，同为第一作者

中图分类号: R575
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出版历程
- 收稿日期: 2020-06-05
- 录用日期: 2020-09-16
- 出版日期: 2021-01-20

Mechanism of Ganshuang granule extract in alleviating N-acetyl-p-aminophenol-induced hepatocellular injury

Qiao WU^{1, 2},
Pengfei YU¹,
Yanzhen BI¹,
Baozeng WANG²,
Zixuan WANG²,
Zhijie LI³,
Yu CHEN^{1
, ,},
Zhongping DUAN^{1
, ,}

1.
a. Intractable Hepatic Diseases and Artificial Liver Treatment & Training Center, b. Beijing Municipal Key Laboratory of Liver Failure and Artificial Liver Treatment Research, Beijing YouAn Hospital, Capital Medical University, Beijing 100069, China
2.
Center of Infectious Diseases, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, China
3.
Center of Hepatobiliary Surgery, The Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China

摘要

摘要: 目的研究肝爽颗粒浸膏减轻对乙酰氨基酚(N-乙酰基-对氨基苯酚，APAP)诱导的肝细胞毒性的能力，以及可能涉及的机制。方法设立5组细胞培养组，分别为正常对照组、APAP损伤组、3组不同肝爽颗粒浸膏浓度的损伤保护组。使用20 mmol/L APAP加入细胞培养液孵育24 h构建造体外药物肝损伤模型，损伤保护组提前加用不同浓度肝爽颗粒浸膏(0.2 μg/ml、1 μg/ml、5 μg/ml)8 h孵育后加入APAP损伤24 h。检测不同组别的肝细胞损伤标志物(ALT、AST、LDH)、线粒体损伤标志物(线粒体膜电位、GDH)、抗氧化及氧化应激标志物(GSH、SOD、MDA、ROS)等。进一步针对实验结果进行机制探讨。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果肝爽颗粒浸膏可以减轻APAP引起的肝细胞毒性，肝爽颗粒浸膏可以提高细胞活力(P＜0.001)，降低上清中AST、ALT、LDH的含量(P值分别为＜0.001、＜0.001、＜0.05);肝爽颗粒浸膏可以抑制APAP诱导的肝细胞氧化应激，肝爽颗粒浸膏组的氧化应激指标ROS、MDA较APAP组下降(P值均＜0.01);肝爽颗粒浸膏可以剂量依赖性减轻APAP诱导的肝细胞线粒体膜电位丢失(P＜0.05)，降低了上清线粒体损伤标志物GDH的含量(P＜0.001);肝爽颗粒浸膏可以抑制CYP2E1/1A2的表达(P值均＜0.05);肝爽颗粒浸膏可以增加肝细胞Ⅱ相酶表达; 肝爽颗粒浸膏可以诱导Nrf2及其下游基因NQO-1及GCLC的表达(P值均＜0.05)。结论肝爽颗粒浸膏可通过两种途径预防APAP诱导肝脏损伤，第一种途径是肝爽颗粒浸膏下调CYP2E1/1A2的表达减少了APAP毒性产物NAPQI的生成; 第二种途径是肝爽颗粒浸膏上调解毒通路的表达，激活Nrf2增加抗氧化酶(SOD、GSH)和Ⅱ相酶的表达，从而加速APAP的无害代谢。
- 药物性肝损伤 /
- 醋氨酚 /
- 肝爽颗粒
Abstract: Objective To investigate the ability of Ganshuang granule (a liver-protecting drug widely used in clinical practice) extract to reduce N-acetyl-p-aminophenol (APAP)-induced hepatotoxicity and possible mechanisms. Methods A total of five cell culture groups were set up in this experiment, i.e., normal control group, APAP injury group, and three injury protection groups treated with different concentrations of Ganshuang granule extract. Then 20 mmol/L APAP was added to the cell culture medium and incubated for 24 hours to establish an in vitro model of drug-induced liver injury, and the injury protection groups were treated with different concentrations of Ganshuang granule extract (0.2, 1, and 5 μg/ml) in advance for 8 hours of incubation before APAP were added for 24 hours. Related markers were measured, including the markers for hepatocellular injury [alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH)], the markers for mitochondrial injury [mitochondrial membrane potential, and glutamate dehydrogenase (GDH)], and antioxidant and oxidative stress markers [glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and reactive oxygen species (ROS)]. Related mechanism was discussed based on the experimental results. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results Ganshuang granule extract alleviated APAP-induced hepatotoxicity, improved cell viability (P < 0.001), and reduced the levels of AST, ALT, and LDH in supernatant (P < 0.001, P < 0.001, and P < 0.05). Ganshuang granule extract inhibited APAP-induced hepatocellular oxidative stress, and compared with the APAP group, the Ganshuang granule extract groups had significant reductions in the oxidative stress indicators ROS and MDA (both P < 0.01). Ganshuang granule extract alleviated the loss of mitochondrial membrane potential induced by APAP (P < 0.05) and reduced the content of the mitochondrial injury marker GDH in supernatant (P < 0.001) in a dose-dependent manner. Ganshuang granule extract inhibited the expression of CYP2E1/1A2 (both P < 0.05) and increased the expression of phase Ⅱ enzymes in hepatocytes. Ganshuang granule extract induced the expression of Nrf2 and its downstream genes NQO-1 and GCLC (all P < 0.05). Conclusion Ganshuang granule extract can prevent APAP-induced hepatocellular injury through two ways. The first way is that Ganshuang granule extract downregulates the expression of CYP2E1/1A2 and thus reduces the production of NAPQI, a toxic product of APAP; the second way is that Ganshuang granule extract upregulates the expression of the detoxification pathway, which can activate Nrf2 to increase the expression of antioxidant enzymes (SOD and GSH) and phase Ⅱ enzymes and thus accelerate the harmless metabolism of APAP.
- Drug-Induced Liver Injury /
- Acetaminophen /
- Ganshuang Granules

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图 1 肝爽浸膏对APAP诱导的线粒体膜电位丢失的影响

注：高内涵监测不同组别0、3 h后线粒体膜电位TRME强度。

下载: 全尺寸图片幻灯片

图 2 肝爽颗粒浸膏对不同组别Ⅱ相酶的影响

注：与APAP组相比, *P＜0.05，**P＜0.001。

下载: 全尺寸图片幻灯片

图 3 肝爽颗粒浸膏诱导Nrf2表达

注：Western Blot检测不同组别Nrf2蛋白水平表达结果。

下载: 全尺寸图片幻灯片

表 1 引物序列

引物名称		序列(5′-3′)
GAPDH	F	GGACTCATGACCACAGTCCA
	R	TCAGCTCAGGGATGACCTTG
UGT1A1	F	CGCCTCTCCAGCCTTCACAAG
	R	GTCAGCACGACGGCCAAGAG
UGT1A3	F	GCCAACAGGAAGCCACTATC
	R	CAGCAATTGCCATAGCTTTC
UGT1A6	F	CCAGTGCCGTATGACCAAGAAGAG
	R	GGAGGCTCTGGCAGTTGATGAAG
UGT2B7	F	GCAGCAGAATACAGCCATTGGATG
	R	GCTGAAGATGCCAGTACAGTCACC
GSTα1	F	TCCAGCTTCCCTCTGCTGAA
	R	GGCTGCCAGGCTGTAGAAAC
GSTM1	F	GCCTGCTCCTGGAATACACAGAC
	R	GCAATGTAGCACAAGATGGCGTTG
SULT1A1	F	TGGTTCAGCACACGTCGTTCAAG
	R	CATCTTCTCCGCATAGTCCGCATC
SULT2A1	F	GAGATTCTCTGCCTGATGCACTCC
	R	ATCACCTTGGCCTTGGAACTGAAG
GCLC	F	ATGGCTTTGAGTGCTGCATCTCC
	R	GGCTCCAGTCCTCGCTCCTC
NQO1	F	GTCGGCAGAAGAGCACTGATCG
	R	ACTCCACCACCTCCCATCCTTTC
Nrf2	F	ACACGGTCCACAGCTCATCAT
	R	TTGGCTTCTGGACTTGGAAC
CYP2E1	F	GCCGACATCCTCTTCCGCAAG
	R	CTGTGGCTTCCAGGCAAGTAGTG
CYP1A2	F	CAACACCTTCTCCATCGCCTCTG
	R	CCACTGACACCACCACCTGATTG

下载: 导出CSV

表 2 肝爽浸膏对APAP诱导的HepG2细胞急性损伤的影响

指标	对照组	APAP组	浸膏0.2 μg/ml组	浸膏1 μg/ml组	浸膏5 μg/ml组	F值	P值
细胞活力(%)	100.00±3.33	37.53±0.51	40.45±0.58	45.02±0.07	43.69±0.80	1668.01	＜0.001
AST(U/ml)	3.80±0.14	7.80±0.14	7.50±0.42	6.70±0.14	6.90±0.14	97.80	＜0.001
ALT(U/ml)	0.55±0.07	1.55±0.07	1.10±0.14	0.85±0.07	0.70±0.00	43.92	＜0.001
LDH(U/ml)	34.25±5.44	56.70±3.25	38.30±5.66	42.35±1.34	35.90±3.25	9.63	＜0.05

下载: 导出CSV

表 3 肝爽浸膏对APAP诱导的氧化应激指标的影响

指标	对照组	APAP组	浸膏0.2 μg/ml组	浸膏1 μg/ml组	浸膏5 μg/ml组	F值	P值
GSH(μmol/g prot)	51.79±9.06	37.14±1.43	47.14±6.23	49.29±4.88	43.21±3.76	3.289	＜0.05
SOD(U/mg prot)	319.74±5.58	245.00±1.77	284.63±0.87	281.25±11.46	281.78±14.68	13.454	＜0.05
ROS(%)	100.00±1.94	277.94±1.41	262.19±1.92	238.47±3.15	231.92±2.80	919.150	＜0.001
MDA(nmol/mg prot)	0.84±0.12	3.70±0.48	1.00±0.12	1.81±0.21	1.08±0.12	66.900	＜0.001

下载: 导出CSV

表 4 肝爽浸膏对APAP诱导的线粒体损伤的影响

指标	对照组	APAP组	浸膏0.2 μg/ml组	浸膏1 μg/ml组	浸膏5 μg/ml组	F值	P值
荧光表达	1.38±0.04	0.28±0.03	0.34±0.03	0.59±0.08	0.76±0.11	35.868	＜0.05
GDH(IU/ml)	43.51±0.37	60.66±1.96	56.26±0.45	54.98±0.45	53.37±0.91	89.590	＜0.001

下载: 导出CSV

表 5 肝爽浸膏对不同组别CYP2E1/1A2 mRNA表达水平的影响

指标	对照组	APAP组	浸膏0.2 μg/ml组	浸膏1 μg/ml组	浸膏5 μg/ml组	F值	P值
CYP2E1	1.00±0.05	1.89±0.31	0.86±0.07	0.64±0.06	0.70±0.09	32.909	＜0.001
CYP1A2	1.00±0.01	1.13±0.11	0.95±0.09	0.67±0.02	0.58±0.05	22.710	＜0.05

下载: 导出CSV

表 6 肝爽浸膏对Nrf2、GCLC及NQO-1 mRNA水平的影响

指标	对照组	APAP组	浸膏0.2 μg/ml组	浸膏1 μg/ml组	浸膏5 μg/ml组	F值	P值
Nrf2	1.00±0.04	0.13±0.01	0.61±0.04	0.75±0.00	0.76±0.15	39.084	＜0.05
NQO-1	1.00±0.08	0.29±0.02	0.46±0.05	0.78±0.06	0.82±0.15	22.456	＜0.05
GCLC	1.00±0.01	0.51±0.09	1.32±0.25	1.29±0.05	1.19±0.18	10.464	＜0.05

下载: 导出CSV

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