鞘氨醇激酶1(Sphk1)抑制剂在肝纤维化大鼠模型中的作用机制

王燕; 马丽; 肖雄; 雷冬梅; 赵奉茹; 郭力源; 王晓忠

doi:10.3969/j.issn.1001-5256.2023.08.018

鞘氨醇激酶1(Sphk1)抑制剂在肝纤维化大鼠模型中的作用机制

DOI: 10.3969/j.issn.1001-5256.2023.08.018

王燕^1, , ,,
马丽²,
肖雄¹,
雷冬梅¹,
赵奉茹¹,
郭力源¹,
王晓忠³

1.
成都中医药大学附属第五人民医院, 成都 611130
2.
中国科学院新疆理化技术研究所, 乌鲁木齐 830000
3.
新疆维吾尔自治区中医医院, 乌鲁木齐 830000

基金项目:

国家自然科学基金 (81860808)

伦理学声明：本研究方案于2020年12月11日经由新疆医科大学实验动物伦理委员会审批，批号：IACUC-20201211-13，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：王燕负责课题设计，资料分析，撰写论文；马丽负责动物实验；肖雄、雷冬梅、赵奉茹、郭力源参与收集数据，整理资料；王晓忠参与实验指导。

详细信息

通信作者:
王燕，wy751105@126.com (ORCID: 0000-0001-9730-8279)

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出版历程
- 收稿日期: 2022-11-25
- 录用日期: 2023-01-19
- 出版日期: 2023-08-20

Mechanism of action of sphingosine kinase 1 inhibitor in a rat model of liver fibrosis

1.
The Fifth People's Hospital Affiliated to Chengdu University of Traditional Chinese Medicine, Chengdu 611130, China
2.
Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830000, China
3.
Traditional Chinese Medicine Hospital of Xinjiang Uygur Autonomous Region, Urumqi 830000, China

Research funding:

National Natural Science Foundtion of China (81860808)

More Information

Corresponding author: WANG Yan, wy751105@126.com (ORCID: 0000-0001-9730-8279)

摘要

摘要: 目的探讨鞘氨醇激酶1(SphK1)抑制剂在不同肝纤维化大鼠模型中的治疗作用。方法 170只SD大鼠随机分为4组：正常组(30只)、HFE组(给予高脂乳剂，45只)、CCl₄组(CCl₄诱导，45只)、CCl₄+HFE组(HFE联合CCl₄，50只)，经病理及实验室检查证实造模成功后，分别给予SphK1抑制剂PF-543，同组别以生理盐水作为对照，分别在第1、7、14天取肝组织进行Masson染色，比较肝纤维化面积占比、透射电镜下观察自噬小体形成情况、Real-time PCR检测肝纤维化及自噬相关标志物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检验。相关性分析采用Spearman相关分析。结果与正常组(0.57±0.13)相比，CCl₄组(6.93±5.81)和HFE+CCl₄组(10.89±2.67)肝纤维化面积占比均升高(P值均＜0.01)。PF-543干预第7天可显著降低CCl₄组及HFE+CCl₄组纤维化病变面积(P值均＜0.01)。与正常组相比，HFE组、CCl₄组和HFE+CCl₄组的SphK1表达水平均明显降低(P值均＜0.01)，HFE+CCl₄组的α平滑肌肌动蛋白、转化生长因子-β、α1-Ⅰ型胶原mRNA表达水平均明显升高(P值均＜0.01)，干预后各指标水平无明显变化(P值均＞0.05)。Atg5、Atg12、Becn1的表达水平与肝纤维化病变面积均呈负相关(r分别为-0.715、-0.640、-0.632，P值均＜0.01)；SphK1的表达水平与Atg5、Atg12、Becn1、Map1lc3a的mRNA表达均呈正相关(r分别为0.603、0.561、0.510、0.498，P值均＜0.01)。电镜示：CCl₄组肝组织轻微水肿，细胞器丰富，轻度肿胀，以粗面内质网扩张为主。该视野可见9个自噬溶酶体结构。在PF-543干预第7天后，未见典型自噬结构。HFE+CCl₄组肝组织轻度水肿，结构不清晰，粗面内质网数量丰富，明显扩张，表面附着核糖体颗粒较少。该视野可见6个自噬溶酶体结构。在PF-543干预第7天后，该视野未见典型自噬结构。结论 PF-543对自噬有显著抑制作用，并同时伴有纤维化面积的减少。提示靶向SphK1，可影响到肝脏的自噬水平，并进而缓解两种肝纤维化模型的纤维化状态。
- 肝纤维化 /
- 鞘氨醇激酶1 /
- 自噬 /
- 模型, 动物
Abstract: Objective To investigate the therapeutic effect of sphingosine kinase 1 (SphK1) inhibitor in different liver fibrosis models. Methods A total of 170 Sprague-Dawley rats were randomly divided into normal group (30 rats), HFE group (45 rats given high-fat emulsion), CCl₄ group (45 rats induced by CCl₄), and CCl₄+HFE group (50 rats given HFE and CCl₄). After successful modeling confirmed by pathology and laboratory examination, the SphK1 inhibitor PF-543 was given, and the rats in the same group were given normal saline as control. Liver tissue samples were collected on days 1, 7, and 14 for Masson staining; the percentage of liver fibrosis area was compared; the formation of autophagosome was observed under a transmission electron microscope; real-time PCR was used to measure the mRNA expression levels of markers associated with liver fibrosis and autophagy. A one-way analysis of variance was used for comparison between multiple groups, and the LSD method was used for further comparison between two groups; a Spearman correlation analysis was also performed. Results Compared with the normal group, the CCl₄ group and the HFE+CCl₄ group had a significant increase in the percentage of liver fibrosis area (6.93±5.81/10.89±2.67 vs 0.57±0.13, both P < 0.01), the CCl₄ group and the HFE+CCl₄ group had a significant reduction in the percentage of liver fibrosis area on day 7 (both P < 0.01). Compared with the normal group, the HFE group, the CCl₄ group, and the HFE+CCl₄ group had a significant reduction in the expression level of SphK1 (all P < 0.01), and the HFE+CCl₄ group had significant increases in the mRNA expression levels of alpha-smooth muscle actin, transforming growth factor-β, and collagen type Ⅰ alpha 1 (all P < 0.01), while there were no significant changes in these indices after intervention (all P > 0.05). The expression levels of Atg5, Atg12, and Becn1 were negatively correlated with the area of liver fibrosis (r=-0.715, -0.640, and -0.632, all P < 0.01), and the expression level of SphK1 was positively correlated with the mRNA expression of Atg5, Atg12, Becn1, and Map1lc3a (r=0.603, 0.561, 0.510, and 0.498, all P < 0.01). Electron microscopy showed that the CCL₄ group had slight edema, abundant organelles, and mild swelling in liver tissue, mainly the expansion of rough endoplasmic reticulum, and nine autolysosome (ASS) structures were seen in this field, while no typical ASS structure was observed on day 7 of PF-543 intervention. The HFE+CCL₄ group had mild edema, unclear structure, and abundant rough endoplasmic reticulum with marked expansion and few ribosome particles attached to its surface in liver tissue, and 6 ASS structures were seen in this field, while no typical ASS structure was observed on day 7 of PF-543 intervention. Conclusion PF-543 significantly inhibits autophagy and is associated with a reduction in fibrosis area. It is suggested that targeting SphK1 can affect the level of liver autophagy, thereby alleviating the state of liver fibrosis in two liver fibrosis models.
- Hepatic Fibrosis /
- Sphingosine Kinase 1 /
- Autophagy /
- Models, Animal

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图 1 各组动物造模后肝脏病理HE染色(×200)

Figure 1. HE staining of liver pathology in each group after modeling (×200)

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图 2 各组予PF-543干预不同时间点纤维化面积比较

Figure 2. Comparison of fibrotic area in each group treated with PF-543 at different time points

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图 3 PF-543抑制剂对各组动物肝纤维化程度的影响(Masson染色，×200)

注：NS，给予生理盐水；黑色箭头，圆形空泡；蓝色箭头，纤维桥接；绿色箭头，假小叶。

Figure 3. Effect of PF-543 inhibitors on the degree of liver fibrosis in animals in each group (Masson staining, ×200)

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图 4 SphK1及纤维化标志物相关基因表达

Figure 4. Expression of SphK1 and related genes of fibrosis markers

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图 5 自噬相关基因表达与Masson面积及SphK1表达量相关性

Figure 5. Correlation analysis of autophagy related gene expression with Masson area and SphK1 expression level

下载: 全尺寸图片幻灯片

图 6 电镜下观察PF-543对各组的影响(×7 000)

Figure 6. Effects of PF-543 on disease model groups observed under electron microscope (×7 000)

下载: 全尺寸图片幻灯片

表 1 各基因PCR引物序列

Table 1. PCR primer sequence of each gene

引物名称	引物序列	产物长度 (bp)
Acta2-F	5′-GGCTGTGATCTCCTTCTG-3′	139
Acta2-R	5′-CATCCACGAAACCACCTAT-3′	139
Atg5-F	5′-ACAACTGAACGGCCTTTC-3′	103
Atg5-R	5′-CTGCGGAAGGACAGACTT-3′	103
Atg12-F	5′-CATTCTACATCCCAAACACATC-3′	116
Atg12-R	5′-AGTTATTGGATTGGCACTGT-3′	116
Becn1-F	5′-TTTCCACCTCTTCTTTGAAC-3′	111
Becn1-R	5′-AGTTGCCGTTGTACTGTT-3′	111
Col1a1-F	5′-CTAACCAAGGCTGCAACC-3′	113
Col1a1-R	5′-GCTGATGTACCAGTTCTTCT-3′	113
Fn1-F	5′-TGAAATGACCACTGCCAAA-3′	137
Fn1-R	5′-GAACCAGCCTACGGATGA-3′	137
Map1lc3a -F	5′-TAAGGAGGTGCAGCAGAT-3′	153
Map1lc3a -R	5′-CGGATGATCTTGACCAACT-3′	153
SphK1-F	5′-CAACACCGATAAGCAGCT-3′	109
SphK1-R	5′-TGGTTGCATAGCCAGGTC-3′	109
TGF-β1-F	5′-CAGAGAAGAACTGCTGTGTA-3′	134
TGF-β1-R	5′-GTCCAGGCTCCAAATGTAG-3′	134
Rat-GAPDH-F	5′-TCTCTTGTGACAAAGTGGACA-3′	128
Rat-GAPDH-R	5′-CCCATTCTCAGCCTTGACTGT-3′	128
注：Col1a1，α1-Ⅰ型胶原；Fn1，纤维连接蛋白；TGF-β，转化生长因子-β。

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