猪去氧胆酸对脂肪变性肝细胞活性的影响及其机制

汪远远; 邹艳; 刘朝霞; 阳学风

doi:10.12449/JCH240212

猪去氧胆酸对脂肪变性肝细胞活性的影响及其机制

DOI: 10.12449/JCH240212

汪远远^{1a, 2},
邹艳^1b,
刘朝霞^{1a, 2},
阳学风^{1a, 2, , ,}

1a.
南华大学附属南华医院消化内科，湖南衡阳 421002
1b.
南华大学附属南华医院手足外科，湖南衡阳 421002
2.
湖南省代谢相关脂肪性肝病临床研究中心，湖南衡阳 421002

基金项目:

湖南省教育厅一般项目 (20C1586);

湖南省科技创新计划项目 (2020SK51910);

湖南省科技创新计划项目 (2021SK51902)

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：汪远远负责实验设计和实施，撰写论文；邹艳负责数据收集与分析；刘朝霞负责图表制作；阳学风指导撰写文章并最后定稿。

详细信息

通信作者:
阳学风， yxf009988@sina.com （ORCID： 0000-0002-3470-0350）

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出版历程
- 收稿日期: 2023-05-08
- 录用日期: 2023-06-09
- 出版日期: 2024-02-19

Effect of hyodeoxycholic acid on the activity of steatosis hepatocytes and its mechanism

Yuanyuan WANG^{1a, 2},
Yan ZOU^1b,
Zhaoxia LIU^{1a, 2},
Xuefeng YANG^{1a, 2
, ,
,}

1a.
Department of Gastroenterology，Nanhua Hospital Affiliated to University of South China，Hengyang，Hunan 421002，China
1b.
Department of Hand and Foot Surgery，Nanhua Hospital Affiliated to University of South China，Hengyang，Hunan 421002，China
2.
Hunan Provincial Clinical Research Center for Metabolic Associated Fatty Liver Disease，Hengyang，Hunan 421002，China

Research funding:

General Project of Hunan Provincial Education Department (20C1586);

Hunan Provincial Science and Technology Innovation Program Project (2020SK51910);

Hunan Provincial Science and Technology Innovation Program Project (2021SK51902)

More Information

Corresponding author: YANG Xuefeng， yxf009988@sina.com （ORCID： 0000-0002-3470-0350）

摘要

摘要: 目的探讨猪去氧胆酸（HDCA）在代谢相关脂肪性肝病（MAFLD）发展中的作用及机制，为进一步阐明MAFLD的发病机制提供新的理论依据。方法 L02肝细胞作为实验细胞，利用棕榈酸诱导L02细胞发生脂肪变性。采用FXR siRNA干扰链技术，构建FXR低表达的肝细胞株。CCK8实验检测HDCA在不同浓度（0、100、200、300、400 μmol/L）和时间（12、24、36、48 h）对L02脂肪变性肝细胞的影响。通过qRT-PCR检测法尼醇X受体（FXR）、增殖细胞核抗原（PCNA）、周期蛋白D1（Cyclin D1）、磷脂酰肌醇-3-激酶（PI3K）和蛋白激酶B（AKT）mRNA表达；Western Blot检测FXR、Cyclin D1、PCNA、PI3K、p-PI3K、AKT和p-AKT蛋白表达。计量资料服从正态分布且方差齐时多组间比较采用单因素方差分析，进一步两两比较采用Tukey HSD检验；服从正态分布但方差不齐时采用Welch方差分析，进一步两两比较采用Games-Howell检验。两组间比较采用成组t检验。结果 CCK8检测结果显示，300 μmol/L HDCA处理的L02细胞和脂肪变性肝细胞活性明显下降（P值均<0.05）；qRT-PCR检测结果显示，FXR mRNA表达增强，PCNA、Cyclin D1、PI3K、AKT的mRNA表达下降，差异均有统计学意义（P值均<0.05）。Western Blot检测结果显示，FXR蛋白表达明显上升（P<0.05）；干扰L02细胞FXR的表达后，PCNA、PI3K、p-PI3K、AKT和p-AKT的蛋白表达均明显增加（P值均<0.05）。结论 HDCA通过上调FXR表达抑制PI3K/AKT信号通路，从而造成脂肪变性肝细胞活性下降。
- 代谢相关脂肪性肝病 /
- 猪去氧胆酸 /
- 法尼醇X受体
Abstract: Objective To investigate the role and mechanism of hyodeoxycholic acid （HDCA） in the progression of metabolic associated fatty liver disease （MAFLD）， and to provide a new theoretical basis for further clarifying the pathogenesis of MAFLD. Methods L02 hepatocytes were used as experimental cells， and palmitic acid was used to induce steatosis in L02 cells. The farnesoid X receptor （FXR） siRNA interference chain technique was used to construct a hepatocyte cell line with low FXR expression. CCK8 assay was used to observe the effect of HDCA on L02 steatosis hepatocytes at different concentrations （0， 100， 200， 300， and 400 μmol/L） and time points （12， 24， 36， and 48 hours）. The method of qRT-PCR was used to measure the mRNA expression levels of FXR， proliferating cell nuclear antigen （PCNA）， Cyclin D1， phosphatidylinositol 3-kinase （PI3K）， and protein kinase-B （AKT）， and Western blot was used to measure the protein expression levels of FXR， Cyclin D1， PCNA， PI3K， phosphorylated PI3K （p-PI3K）， AKT， and phosphorylated （p-AKT）. A one-way analysis of variance was used for comparison of normally distributed continuous data with homogeneity of variance between multiple groups， and the Tukey HSD test was used for further comparison between two groups； the Welch analysis of variance was used for comparison of normally distributed continuous data with heterogeneity of variance between multiple groups， and the Games-Howell test was used for further comparison between two groups. The independent-samples t test was used for comparison between two groups. Results CCK8 assay showed a significant reduction in the viability of L02 cells and steatosis hepatocytes treated by 300 μmol/L HDCA （P<0.05）， and qRT-PCR showed a significant increase in the mRNA expression level of FXR and significant reductions in the mRNA expression levels of PCNA， Cyclin D1， PI3K， and AKT （all P<0.05）. Western blot showed a significant increase in the protein expression level of FRX （P<0.05）， and after interference of FXR expression in L02 cells， there were significant increases in the protein expression levels of PCNA， PI3K， p-PI3K， AKT， and p-AKT （all P<0.05）. Conclusion HDCA inhibits the PI3K/AKT signaling pathway by upregulating FXR expression， thereby inducing a reduction in the viability of steatosis hepatocytes.
- Metabolism-Associated Fatty Liver Disease /
- Hyodeoxycholic Acid /
- Farnesoid X Receptor

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图 1 油红O染色结果（×20）

注： a，PA诱导前油红O染色；b，PA诱导后24 h油红O染色。

Figure 1. Results of the oil red O staining

下载: 全尺寸图片幻灯片

图 2 PA诱导L02细胞后甘油三酯水平随时间的变化

Figure 2. Changes in triglyceride levels over time after PA induction in L02 cells

下载: 全尺寸图片幻灯片

图 3 HDCA对正常肝细胞及脂肪变性肝细胞活性的影响

注： a，不同浓度HDCA对L02细胞活性的影响；b，不同浓度HDCA对PA诱导的脂肪变性肝细胞活性的影响；c，HDCA（300 μmol/L）不同时间对PA诱导的脂肪变性肝细胞活性的影响。

Figure 3. Effect of HDCA on the activity of normal hepatocytes and steatosis hepatocytes

下载: 全尺寸图片幻灯片

图 4 HDCA对脂肪变性肝细胞FXR表达的影响

注： a，FXR蛋白印迹图；b，FXR蛋白相对表达量。

Figure 4. Effect of HDCA on the expression of FXR in steatotic hepatocytes

下载: 全尺寸图片幻灯片

图 5 FXR siRNA干扰效果及HDCA对脂肪变性肝细胞FXR表达的影响

注： a，3条FXR siRNA干扰链对脂肪变性肝细胞中FXR mRNA表达的干扰效果；b，FXR siRNA 3对HDCA刺激的干扰效果。

Figure 5. Effect of FXR siRNA interference and the effect of HDCA on FXR expression in lipid-denatured hepatocytes

下载: 全尺寸图片幻灯片

图 6 抑制FXR表达后脂肪变性肝细胞FXR-PI3K/AKT通路关键分子及PCNA和Cyclin D1蛋白表达的变化

注： a，FXR、PCNA、Cyclin D1、PI3K、p-PI3K、AKT和p-AKT的蛋白印迹图；b，FXR蛋白的相对表达水平；c，PCNA蛋白的相对表达水平；d，Cyclin D1蛋白的相对表达水平；e，PI3K蛋白的相对表达水平；f，p-PI3K蛋白的相对表达水平；g，AKT蛋白的相对表达水平；h，p-AKT蛋白的相对表达水平。

Figure 6. Changes in the expression of key molecules of FXR-PI3K/AKT pathway and PCNA and Cyclin D1 proteins in steatosis hepatocytes after inhibition of FXR expression

下载: 全尺寸图片幻灯片

表 1 qRT-PCR引物序列

Table 1. qRT-PCR primer sequences

引物	序列（5'-3'）
FXR	上游：AACCATACTCGCAATACAGCAA
	下游：ACAGCTCATCCCCTTTGATCC
PCNA	上游：CCTGCTGGGATATTAGCTCCA
	下游：CAGCGGTAGGTGTCGAAGC
Cyclin D1	上游：GCTGCGAAGTGGAAACCATC
	下游：CCTCCTTCTGCACACATTTGAA
PI3K	上游：TATTTGGACTTTGCGACAAGACT
	下游：TCGAACGTACTGGTCTGGATAG
AKT-F	上游：AGCGACGTGGCTATTGTGAAG
	下游：GCCATCATTCTTGAGGAGGAAGT

下载: 导出CSV

表 2 HDCA对脂肪变性肝细胞FXR、PI3K、AKT、PCNA和Cyclin D1 mRNA表达的影响

Table 2. Effect of HDCA on the mRNA expression of FXR， PI3K， AKT， PCNA， and Cyclin D1 in steatotic hepatocytes

指标	M组	M+HDCA组	t值	P值
FXR mRNA	0.485±0.162	1.010±0.013	5.576	0.005
PCNA mRNA	1.710±0.052	1.034±0.054	-15.679	<0.001
Cyclin D1 mRNA	1.435±0.124	1.041±0.053	-5.083	0.007
PI3K mRNA	1.951±0.500	1.008±0.009	-3.266	0.031
AKT mRNA	2.373±0.316	1.030±0.050	-7.266	0.002

下载: 导出CSV

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