二甲双胍对布加综合征小鼠模型肝纤维化的影响

杨净; 李素新; 张跃辉; 李路豪; 刘兆臣; 沈东启; 党晓卫

doi:10.3969/j.issn.1001-5256.2022.09.017

二甲双胍对布加综合征小鼠模型肝纤维化的影响

DOI: 10.3969/j.issn.1001-5256.2022.09.017

郑州大学第一附属医院肝胆胰外科，郑州 450052

基金项目:

河南省医学科技攻关计划省部共建项目 (SB201901003)

伦理学声明：本研究方案于2019年7月10日经由郑州大学第一附属医院实验动物伦理委员会审批，批号为2019-KY-144，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、伦理委员会成员以及与公开研究成果有关的利益冲突。

作者贡献声明：杨净参与课题设计，实施课题，撰写论文；张跃辉、沈东启参与课题实施；李路豪、刘兆臣参与课题设计，修改论文；李素新、党晓卫负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
党晓卫，dangxw1001@163.com

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出版历程
- 收稿日期: 2022-01-20
- 录用日期: 2022-03-02
- 出版日期: 2022-09-20

Effect of metformin on liver fibrosis in a mouse model of Budd-Chiari syndrome

Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China

Research funding:

Henan Province Medical Science and Technology Research Plan Joint Provincial and Ministry Project (SB201901003)

More Information

Corresponding author: DANG Xiaowei, dangxw1001@163.com(ORCID: 0000-0001-7940-6385)

摘要

摘要: 目的探讨二甲双胍对于布加综合征小鼠模型肝脏纤维化的影响及其机制。方法 C57雄性小鼠30只随机分为4组：假手术组(SHAM组)6只，假手术+二甲双胍组(SHAM+M组)5只，布加综合征模型组(BCS组)10只，布加综合征模型+二甲双胍组(BCS+M组)9只。模型组行下腔静脉部分结扎术，SHAM组不结扎，二甲双胍组造模同时饮水中给予0.1%二甲双胍。6周后处死小鼠取材，HE染色、天狼猩红染色观察小鼠肝脏组织病理和胶原沉积变化，免疫组化检测α-平滑肌肌动蛋白(α-SMA)、纤维蛋白原(Fibrinogen)表达情况，实时荧光定量PCR检测缺氧诱导因子-1α(HIF-1α)、Ⅰ型胶原(Collagen1)的mRNA表达，Western Blot检测HIF-1α、血管内皮生长因子(VEGF)、Fibrinogen、α-SMA、Collagen1蛋白的相对表达量。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SHAM组相比，病理染色结果显示BCS组肝脏明显纤维化，中央静脉旁肝细胞排列紊乱，肝窦扩张并伴有红细胞淤积和少量炎症细胞浸润，胶原沉积。BCS组HIF-1α、Collagen1 mRNA表达增加，α-SMA、Collagen1、HIF-1α、VEGF、Fibrinogen蛋白表达明显增加(P值均＜0.05)；与BCS组相比，BCS+M组肝脏纤维化减轻，红细胞淤积减少，胶原沉积减少，HIF-1α、Collagen1 mRNA表达降低，α-SMA、Collagen1、HIF-1α、VEGF、Fibrinogen蛋白表达降低(P值均＜0.05)。结论二甲双胍能够改善由布加综合征所导致的淤血性肝纤维化，其机制可能为减少肝窦内微血栓，抑制HIF-1α/VEGF通路来发挥作用。
- Budd-Chiari综合征 /
- 肝硬化 /
- 二甲双胍 /
- 小鼠, 近交C57BL
Abstract: Objective To investigate the effect of metformin on liver fibrosis in a mouse model of Budd-Chiari syndrome and its mechanism. Methods A total of 30 male C57 mice were randomly divided into sham-operation group (SHAM group) with 6 mice, sham operation+ metformin group (SHAM+M group) with 5 mice, Budd-Chiari model group (BCS group) with 10 mice, and Budd-Chiari model+metformin group (BCS+M group) with 9 mice. The mice in the model group were treated with partial ligation of the inferior vena cava, those in the SHAM group were not treated with ligation, and those in the metformin group were given 0.1% metformin in drinking water besides modeling. The mice were sacrificed after 6 weeks. HE staining and picrosirius red staining were used to observe liver histopathology and collagen deposition; immunohistochemistry was used to measure the expressions of α-smooth muscle actin (α-SMA) and fibrinogen; quantitative real-time PCR was used to measure the mRNA expression of hypoxia-inducible factor 1α (HIF-1α) and type Ⅰ collagen (collagen 1), and Western blot was used to measure the relative protein expression levels of HIF-1α, vascular endothelial growth factor (VEGF), fibrinogen, α-SMA, and collagen 1. 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 Pathological staining showed that compared with the SHAM group, the BCS group had significant liver fibrosis, disordered arrangement of hepatocytes near the central vein, sinusoidal expansion with red blood cell deposition and a small amount of inflammatory cell infiltration, and collagen deposition. The BCS group had significant increases in the mRNA expression levels of HIF-1α and collagen 1 and the protein expression levels of α-SMA, collagen 1, HIF-1α, VEGF, and fibrinogen (all P < 0.05); compared with the BCS group, the BCS+M group had significant alleviation of liver fibrosis, red blood cell deposition, and collagen deposition and significant reductions in the mRNA expression levels of HIF-1α and collagen 1 and the protein expression levels of α-SMA, collagen 1, HIF-1α, VEGF, and fibrinogen (all P < 0.05). Conclusion Metformin can improve congestive liver fibrosis caused by Budd-Chiari syndrome, possibly by reducing microthrombus in hepatic sinusoid and inhibiting the HIF-1α/VEGF pathway.
- Budd-Chiari Syndrome /
- Liver Cirrhosis /
- Metformin /
- Mice, Inbred C57BL

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图 1 小鼠肝脏组织标本

注：a, 肝脏组织标本(造模后6周); b, 各组小鼠肝指数(肝质量/体质量)。

Figure 1. Liver samples of mouse

下载: 全尺寸图片幻灯片

图 2 各组小鼠肝脏HE染色情况

Figure 2. HE staining of livers of mice in each group

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图 3 小鼠肝脏胶原及α-SMA表达情况

注：a, 天狼猩红染色(×100);b，α-SMA免疫组化染色(×200);c，Western Blot结果; d，Collagen1 mRNA相对表达水平; e，各组天狼猩红染色面积比较；f，α-SMA免疫组化染色面积比较；g，Collagen1蛋白相对表达水平；h，α-SMA蛋白相对表达水平。

Figure 3. Expression of collagen and α-SMA in mouse liver

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图 4 Fibrinogen表达情况

注：a, Fibrinogen免疫组化染色(×200);b, Western Blot结果; c, Fibrinogen免疫组化染色面积；d, Fibrinogen蛋白相对表达水平。

Figure 4. Expression of Fibrinogen

下载: 全尺寸图片幻灯片

图 5 小鼠肝脏HIF-1α和VEGF表达情况

注：a, Western Blot结果; b, HIF-1α mRNA相对表达水平；c, HIF-1α蛋白相对表达水平；d, VEGF蛋白相对表达水平。

Figure 5. Expression of HIF-1α and VEGF in mouse liver

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表 1 qPCR引物序列

Table 1. The primers used for qPCR analysis

引物名称	序列(5′-3′)
GAPDH F	AGGTCGGTGTGAACGGATTTG
GAPDH R	TGTAGACCATGTAGTTGAGGTCA
Collagen1 F	GCTCCTCTTAGGGGCCACT
Collagen1 R	CCACGTCTCACCATTGGGG
HIF-1α F	ACCTTCATCGGAAACTCCAAAG
HIF-1α R	ACTGTTAGGCTCAGGTGAACT

下载: 导出CSV

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