基于Keap1/Nrf2信号通路探讨丹葛解酲汤对酒精性肝病大鼠模型的作用机制

王铭; 徐建虎; 马丽

doi:10.3969/j.issn.1001-5256.2023.05.018

基于Keap1/Nrf2信号通路探讨丹葛解酲汤对酒精性肝病大鼠模型的作用机制

DOI: 10.3969/j.issn.1001-5256.2023.05.018

王铭¹,
徐建虎^{1, 2},
马丽^{1, 2, , ,}

1.
宁夏医科大学中医学院，银川 750004
2.
宁夏少数民族医药现代化教育部重点实验室，银川 750004

基金项目:

宁夏自然科学基金 (2021AAC03146);

宁夏医科大学校级特殊人才启动项目 (XT2020027)

伦理学声明：本研究方案于2022年4月7日经由宁夏医科大学实验动物中心实验动物伦理委员会审批，批号：IACUC-NYLAC-2021-119，符合实验室动物管理与使用准则。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：王铭负责实验操作，整理数据，撰写文章；徐建虎负责指导实验和写作，分析数据；马丽负责实验设计，数据分析，拟定写作思路，修改文章及定稿。

详细信息

通信作者:
马丽，mali3549@163.com (ORCID: 0000-0002-6646-3438)

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出版历程
- 收稿日期: 2022-09-25
- 录用日期: 2022-11-25
- 出版日期: 2023-05-20

Mechanism of action of Dange Jiecheng decoction in a rat model of alcoholic liver disease based on the Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 signaling pathway

Ming WANG¹,
Jianhu XU^{1, 2},
Li MA^{1, 2
, ,
,}

1.
School of Traditional Chinese Medicine, Ningxia Medical University, Yinchuan 750004, China
2.
Key Laboratory of Ningxia Minority Medicine Modernization, Ministry of Education, Yinchuan 750004, China

Research funding:

Natural Science Foundation of Ningxia (2021AAC03146);

University Level Special Talents Start-up Project of Ningxia Medical University (XT2020027)

More Information

Corresponding author: MA Li, mali3549@163.com (ORCID: 0000-0002-6646-3438)

摘要

摘要: 目的观察丹葛解酲汤对酒精性肝病(ALD)大鼠模型的治疗作用，探讨丹葛解酲汤抗氧化应激的作用机制。方法将96只SD大鼠随机分为空白组(n=13)和ALD组(n=83)，ALD组采用白酒灌胃法建立ALD模型。随后将存活ALD组大鼠随机分为模型组、丹葛解酲汤高剂量组(24 g/kg)、丹葛解酲汤低剂量组(6 g/kg)及益肝灵片组(21 mg/kg)，每组各17只，空白组和模型组给予生理盐水灌胃，其余组分别灌胃给予相应的药物，共干预4周。采用HE染色法观察大鼠肝组织的病理变化；Western Blot法检测肝脏组织Kelch样ECH相关蛋白1(Keap1)、核转录因子E2相关因子2(Nrf2)、血红素氧合酶1(HO-1)的蛋白含量；实时荧光定量PCR检测肝组织Keap1、HO-1 mRNA表达水平。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。结果与空白组相比，模型组肝细胞排列紊乱，出现肝细胞坏死，有大量炎性细胞浸润，并伴有大量的脂滴空泡；Keap1蛋白、Keap1 mRNA表达水平均明显上升(P值均＜0.05)，Nrf2蛋白、HO-1蛋白、HO-1 mRNA表达水平均明显下降(P值均＜0.05)。与模型组相比，丹葛解酲汤高剂量组、丹葛解酲汤低剂量组及益肝灵片组肝细胞排列变整齐，坏死及炎性细胞减少，偶见或无脂滴空泡；Keap1蛋白、Keap1 mRNA表达水平显著降低(P值均＜0.05)，Nrf2蛋白、HO-1蛋白、HO-1 mRNA表达水平显著上升(P值均＜0.05)。结论丹葛解酲汤可能通过调节Keap1/Nrf2信号通路，促使Nrf2入核，上调HO-1的表达，减轻氧化应激反应，进而对ALD大鼠发挥保护作用。
- 肝疾病, 酒精性 /
- Kelch样ECH相关蛋白1 /
- NF-E2相关因子2 /
- 丹葛解酲汤 /
- 大鼠, Sprague-Dawley
Abstract: Objective To investigate the therapeutic effect of Dange Jiecheng decoction on a rat model of alcoholic liver disease (ALD) and the anti-oxidative stress mechanism of Dange Jiecheng decoction. Methods A total of 96 Sprague-Dawley rats were randomly divided into blank group with 13 rats and ALD group with 83 rats, and the rats in the ALD group were given liquor by gavage to establish a model of ALD. Then the ALD group was randomly divided into model group, high-dose Dange Jiecheng decoction group (24 g/kg), low-dose Dange Jiecheng decoction group (6 g/kg), and Yiganling tablet group (21 mg/kg), with 17 rats in each group. The rats in the blank group and the model group were given normal saline by gavage, and those in the other groups were given corresponding drugs by gavage, for 4 consecutive weeks. HE staining was used to observe the pathological changes of liver tissue; Western blot was used to measure the contents of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in liver tissue; quantitative real-time PCR was used to measure the mRNA expression levels of Keap1 and HO-1 in liver tissue. 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 Compared with the blank group, the model group had disordered arrangement of hepatocytes with necrosis, massive inflammatory cell infiltration, and a large number of lipid droplet vacuoles, significant increases in the protein and mRNA expression levels of Keap1 (P < 0.05), and significant reductions in the protein expression levels of Nrf2 and HO-1 and the mRNA expression level of HO-1 (P < 0.05). Compared with the model group, the high- and low-dose Dange Jiecheng decoction groups and the Yiganling tablet group had ordered arrangement of hepatocytes, reductions in hepatocyte necrosis and inflammatory cells, and occasional lipid droplet vacuoles, as well as significant reductions in the protein and mRNA expression levels of Keap1 (P < 0.05) and significant increases in the protein expression levels of Nrf2 and HO-1 and the mRNA expression level of HO-1 (P < 0.05). Conclusion By regulating the Keap1/Nrf2 signaling pathway, Dange Jiecheng decoction can promote the nuclear import of Nrf2, upregulate the expression of HO-1, and alleviate oxidative stress response, thereby exerting a protective effect on ALD rats.
- Liver Diseases, Alcoholic /
- Kelch-Like ECH-Associated Protein 1 /
- NF-E2-Related Factor 2 /
- Dange Jiecheng Decoction /
- Rats, Sprague-Dawley

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图 1 各组大鼠肝脏组织病理学变化(HE染色，×200)

注：a，空白组；b，模型组；c，益肝灵片组；d，中药低剂量组；e，中药高剂量组。

Figure 1. Pathological changes in liver tissue of rats in each group (HE, ×200)

下载: 全尺寸图片幻灯片

图 2 各组大鼠肝组织Keap1、Nrf2、HO-1蛋白表达量

注：a，空白组；b，模型组；c，益肝灵片组；d，中药低剂量组；e，中药高剂量组。

Figure 2. The protein expression of Keap1, Nrf2 and HO-1 in liver tissue of rats in each group was detected

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequence

基因	上游引物(5′-3′)	下游引物(5′-3′)	产物长度(bp)
Keap1	CCTCCTCCAGCCCAGTCTTT	GCCGTGTAGGCGAACTCAAT	127
HO-1	CGCATGAACACTCTGGAGATG	TGTGAGGGACTCTGGTCTTTGT	143
β-actin	CGTAAAGACCTCTATGCCAACA	TAGGAGCCAGGGCAGTAATC	154

下载: 导出CSV

表 2 各组大鼠Keap1、Nrf2、HO-1蛋白表达量比较

Table 2. Comparison of Keap1、Nrf2、HO-1 protein expression in each group

组别	动物数(只)	Keap1	Nrf2	HO-1
空白组	3	0.38±0.03	0.88±0.05	1.84±0.10
模型组	3	1.19±0.04¹⁾	0.30±0.03¹⁾	0.56±0.11¹⁾
益肝灵片组	3	0.44±0.04²⁾	0.10±0.09²⁾	1.80±0.07²⁾
中药低剂量组	3	0.72±0.09²⁾	0.59±0.03²⁾	1.14±0.09²⁾
中药高剂量组	3	0.45±0.04²⁾	0.92±0.06²⁾	1.46±0.09²⁾
F值		118.374	64.316	96.754
P值		＜0.01	＜0.01	＜0.01
注：与空白组比较，1)P＜0.05；与模型组比较，2)P＜0.05。

下载: 导出CSV

表 3 各组大鼠Keap1、HO-1 mRNA表达水平比较

Table 3. Comparison of Keap1 and HO-1 mRNA expression levels in rats of each group

组别	动物数(只)	Keap1 mRNA	HO-1 mRNA
空白组	3	1.01±0.12	1.09±0.09
模型组	3	6.70±1.19¹⁾	0.27±0.06¹⁾
益肝灵片组	3	1.83±0.09²⁾	0.73±0.09²⁾
中药低剂量组	3	4.37±1.29²⁾	0.48±0.06²⁾
中药高剂量组	3	2.20±0.16²⁾	0.61±0.11²⁾
F值		25.493	39.035
P值		＜0.01	＜0.01
注：与空白组比较，1)P＜0.05；与模型组比较，2)P＜0.05。

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