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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
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。
Mechanism of Ganshuang granule extract in alleviating N-acetyl-p-aminophenol-induced hepatocellular injury
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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。 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. -
Key words:
- Drug-Induced Liver Injury /
- Acetaminophen /
- Ganshuang Granules
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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 
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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
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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
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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
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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
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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
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