舒肝宁注射液对于氯丙嗪引起的新型组织工程肝构建的胆汁淤积型药物性肝损伤模型的影响

黄龙; 陈煜; 吴桥; 段钟平

doi:10.3969/j.issn.1001-5256.2022.03.018

舒肝宁注射液对于氯丙嗪引起的新型组织工程肝构建的胆汁淤积型药物性肝损伤模型的影响

DOI: 10.3969/j.issn.1001-5256.2022.03.018

黄龙^{1a, 1b},
陈煜^{1a, 1b},
吴桥^{1a, 1b, 2, , ,},
段钟平^{1a, 1b, , ,}

1a.
首都医科大学附属北京佑安医院肝病中心四科，北京 100069
1b.
首都医科大学附属北京佑安医院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

基金项目:

大数据精准医疗高精尖创新中心 (1212040205);

北京市自然科学基金 (7222094);

首都卫生发展科研专项项目 (2021-1G-2181)

伦理学声明：本研究方案于2020年6月5日经由北京佑安医院实验动物伦理委员会审批通过，批号：AEEI-2020-076，符合实验室动物管理与使用准则。

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：黄龙负责撰写论文；吴桥负责课题设计，修改论文；陈煜、段钟平负责提供写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
吴桥，E-mail: wuqiao322@163.com

段钟平，E-mail: duan@ccmu.edu.cn

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出版历程
- 收稿日期: 2021-07-13
- 录用日期: 2021-08-12
- 出版日期: 2022-03-20

Effect of Shuganning injection on the model of cholestasis - type chlorpromazine - induced liver injury constructed by a new tissue - engineered liver

Long HUANG^{1a, 1b},
Yu CHEN^{1a, 1b},
Qiao WU^{1a, 1b, 2
, ,
,},
Zhongping DUAN^{1a, 1b
, ,
,}

1a.
Fourth Department of Liver Disease Center, Beijing YouAn Hospital, Capital Medical University, Beijing 100069, China
1b.
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

Research funding:

Big Data Precision Medicine Advanced Innovation Center (1212040205);

Beijing Natural Science Foundation (7222094);

Capital Health Development Scientific Research Special Project (2021-1G-2181)

More Information

Corresponding author: WU Qiao, wuqiao322@163.com(ORCID: 0000-0001-7667-5002); DUAN Zhongping, duan@ccmu.edu.cn(ORCID: 0000-0002-8455-0426)

摘要

摘要: 目的研究舒肝宁注射液(SGN)减轻药物引起的胆汁淤积症以及可能涉及的机制。方法将Sprague-Dawley大鼠肝脏去细胞化制为肝脏胶原支架，用人HepG2细胞对支架再细胞化，从而获得组织工程肝(正常对照组)，加入10 μmol/L浓度的氯丙嗪(CPZ)与胆汁酸盐混合物灌注组织工程肝建立药物性胆汁淤积模型(CPZ损伤组)，该模型进一步用舒肝宁注射液(10³倍稀释)处理后为损伤保护组(CPZ+SGN)。随后检测不同组别的肝细胞损伤标志物(ALT、AST、LDH、ALP)、抗氧化及氧化应激标志物(GSH、MDA、SOD、ROS)，比较正常组、CPZ组和SGN组肝脏胆汁酸盐代谢相关酶类mRNA以及Western Blot表达水平; 比较正常组、CPZ组和SGN组肝脏胆汁淤积相关酶类mRNA以及Western blot表达水平。并行HE染色评估肝脏病理学。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 CPZ+SGN组ALT、AST、LDH、ALP肝细胞损伤标记指标较CPZ损伤组显著降低(P值均 < 0.000 1)，CPZ+SGN组GSH、SOD氧化应激标志物较CPZ组升高(P值分别为 < 0.000 1、 < 0.001)，MDA和ROS指标较CPZ组降低(P值分别为 < 0.000 1、 < 0.001)。CPZ+SGN组较CPZ组肝细胞的胆固醇7α-羟化酶、胆固醇12α羟化酶的mRNA表达明显降低(P值均 < 0.001)，法尼醇X受体、小异二聚体伴侣、胆盐输出泵及和多药耐药相关蛋白2的mRNA表达水平显著升高(P值分别为 < 0.000 1、 < 0.01、 < 0.000 1、 < 0.000 1)。HE染色结果显示CPZ+SGN组肝细胞的损伤明显减少，细胞的数量相对于损伤组有明显的提高。结论 SGN可以减轻CPZ引起的药物性胆汁淤积肝损伤，其主要保护机制是通过激活肝细胞的法尼醇X受体，增加小异二聚体伴侣表达发挥调控胆汁酸盐平衡的保护作用，同时抑制胆固醇7α-羟化酶和胆固醇12α羟化酶等减少疏水性胆汁酸合成，上调胆盐输出泵、多药耐药相关蛋白2的表达促进胆汁酸盐的排出。
- 化学性与药物性肝损伤 /
- 胆汁淤积 /
- 舒肝宁注射液 /
- 大鼠, Sprague-Dawley
Abstract: Objective To investigate the effect of Shuganning injection (SGN) in alleviating drug-induced cholestasis and the possible mechanisms involved. Methods The liver of Sprague-Dawley rats was decellularized to prepare collagen scaffolds, and then the scaffolds were recellularized with human HepG2 cells to obtain the tissue-engineered liver (normal control group). The tissue-engineered liver was perfused with 10 μmol/L chlorpromazine (CPZ) and bile salt mixture to establish a model of drug-induced cholestasis (CPZ group), and the model was further treated with Shuganning injection (10³-fold dilution) as the injury protection group (SGN+CPZ group). The markers for hepatocellular injury [alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP)] and the antioxidant and oxidative stress markers [glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS)] were measured for all groups, and the normal control group, the CPZ group, and the SGN+CPZ group were compared in terms of the mRNA and protein expression levels of the enzymes associated with liver bile salt metabolism and the enzymes associated with hepatic cholestasis. HE staining was performed to observe liver pathology. 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 CPZ group, the SGN+CPZ group had significant reductions in the markers for hepatocellular injury ALT, AST, LDH, and ALP (all P < 0.000 1), significant increases in the oxidative stress markers GSH and SOD (P < 0.000 1 and P < 0.001), and significant reductions in the markers MDA and ROS (P < 0.000 1 and P < 0.001). Compared with the CPZ group, the SGN+CPZ group had significant reductions in the mRNA expression levels of cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CPY8B1) in hepatocytes (all P < 0.001) and significant increases in the mRNA expression levels of farnesoid X receptor (FXR), small heterodimeric partner (SHP), bile salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2) (P < 0.000 1, P < 0.01, P < 0.000 1, and P < 0.000 1). HE staining showed that compared with the CPZ group, the SGN+CPZ group had a significant reduction in hepatocyte injury and a significant increase in the number of cells. Conclusion Shuganning injection can alleviate drug-induced cholestatic liver injury caused by chlorpromazine, and it exerts a protective effect by activating FXR in hepatocytes and increasing the expression of SHP to regulate bile salt balance. It also inhibits CYP7A1 and CYP8B1 to reduce the synthesis of hydrophobic bile acids and upregulates the expression of BSEP and MRP2 to promote the excretion of bile salts.
- Chemical and Drug Induced Liver Injury /
- Cholic Acids /
- Shuganning injection /
- Rats, Sprague-Dawley

HTML全文

图 1 制备CPZ所致胆汁淤积型肝损伤及药物干预流程

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图 2 DILI模型的建立

注：a，CPZ损伤组；b，正常对照组。

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图 3 细胞毒性和保护作用

注：a，不同SGN浓度对细胞的毒性；b，不同浓度SGN对细胞的保护作用。S3，SNG稀释10³倍；S4，SGN稀释10⁴倍；S5，SGN稀释10⁵倍；S6，SGN稀释10⁶倍；S7，SGN稀释10⁷倍，S8，SGN稀释10⁸倍。与CPZ损伤组比较，*P < 0.001，**P < 0.000 1。

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图 4 SGN对氧化应激的保护作用

注：与CPZ损伤组比较，*P < 0.001, **P < 0.000 1。

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图 5 不同组别组织工程肝脏的HE染色结果(×10)

注：a，正常组；b，CPZ损伤组；c，SGN+CPZ保护组。

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图 6 不同组别组织工程肝脏的上清生化检测结果

注：与CPZ损伤组比较，**P < 0.000 1。

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图 7 不同组别的CYP7A1和CYP8B1的基因及蛋白表达水平

注：与CPZ损伤组比较，*P < 0.001, **P < 0.000 1。

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图 8 FXR、SHP两种核受体的激活增加了BSEP、MRP2的表达

注：与CPZ损伤组比较，*P < 0.01, **P < 0.000 1。

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图 9 舒肝宁保护机制的示意图

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表 1 1000×的混合胆汁酸盐配方表

混合胆汁酸盐	体积
胆酸	410 μL
鹅去氧胆酸	640 μL
脱氧胆酸	480 μL
石胆酸	8 μL
熊去氧胆酸	14 μL
甘氨鹅脱氧胆酸	160 μL
总计	1712 μL

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表 2 PCR引物序列

引物名称		序列(5′-3′)
GAPDH	F	GGACTCATGACCACAGTCCA
	R	TCAGCTCAGGGATGACCTTG
SHP	F	CTCGGTTTGCATACAGTGTTTGAC
	R	GCATATTGGCCTGGAGGTTTT
CYP7A1	F	CAGAACTGAATGACCTGCCA
	R	GGTGCAAAGTGAAATCCTCC
CYP8B1	F	ATGAAGGCTGTGCGAGAG
	R	TCTCTTCCATCACGCTGTC
BSEP	F	TGAGCCTGGTCATCTTGTG
	R	TCCGTAAATATTGGCTTTCTG
MRP2	F	ACAGAGGCTGGTGGCAACC
	R	ACCATTACCTTGTCACTGTCCATGA
FXR	F	ACTGACCTGTGAGGGGTGTA
	R	TGCCCCCGTTTTTACACTTG
注：CYP7A1，胆固醇7α-羟化酶；CYP8B1，甾醇12α-羟化酶；SHP，小异二聚体伴侣。

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