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烟酰胺单核苷酸对Huh7细胞胆固醇代谢调节作用的研究
作者:
作者单位:

(南华大学衡阳医学院心血管疾病研究所 动脉硬化学湖南省重点实验室 湖南省动脉硬化性疾病国际科技创新合作基地,湖南省衡阳市 421001)

作者简介:

刘文凤,硕士研究生,研究方向为动脉粥样硬化病因发病学与防治基础,E-mail为654045281@qq.com。

基金项目:

湖南省自然科学基金(2017JJ3277)；湖南省卫健委2020年重点指导课题(20201905)


Effect of nicotinamide mononucleotide on cholesterol metabolism in Huh7 cells
Author:
  • LIU Wenfeng

    LIU Wenfeng

    Institute of Cardiovascular Disease & Key Laboratory for Arteriosclerology of Hunan Province & Hunan International Scientific and Technological Cooperation Base of Arteriosclerotic Disease, Hengyang Medical College, University of South China, Hengyang, Hunan 421001, China
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  • LEI Xiaocan

    LEI Xiaocan

    Institute of Cardiovascular Disease & Key Laboratory for Arteriosclerology of Hunan Province & Hunan International Scientific and Technological Cooperation Base of Arteriosclerotic Disease, Hengyang Medical College, University of South China, Hengyang, Hunan 421001, China
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  • XIANG Qiong

    XIANG Qiong

    Institute of Cardiovascular Disease & Key Laboratory for Arteriosclerology of Hunan Province & Hunan International Scientific and Technological Cooperation Base of Arteriosclerotic Disease, Hengyang Medical College, University of South China, Hengyang, Hunan 421001, China
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  • LIAO Wei

    LIAO Wei

    Institute of Cardiovascular Disease & Key Laboratory for Arteriosclerology of Hunan Province & Hunan International Scientific and Technological Cooperation Base of Arteriosclerotic Disease, Hengyang Medical College, University of South China, Hengyang, Hunan 421001, China
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  • XIA Mengdie

    XIA Mengdie

    Institute of Cardiovascular Disease & Key Laboratory for Arteriosclerology of Hunan Province & Hunan International Scientific and Technological Cooperation Base of Arteriosclerotic Disease, Hengyang Medical College, University of South China, Hengyang, Hunan 421001, China
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  • PENG Juan

    PENG Juan

    Institute of Cardiovascular Disease & Key Laboratory for Arteriosclerology of Hunan Province & Hunan International Scientific and Technological Cooperation Base of Arteriosclerotic Disease, Hengyang Medical College, University of South China, Hengyang, Hunan 421001, China
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Affiliation:

Institute of Cardiovascular Disease & Key Laboratory for Arteriosclerology of Hunan Province & Hunan International Scientific and Technological Cooperation Base of Arteriosclerotic Disease, Hengyang Medical College, University of South China, Hengyang, Hunan 421001, China)

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    摘要:

    目的 探讨烟酰胺单核苷酸(NMN)对Huh7细胞胆固醇的调节作用及分子机制。方法 用不同浓度(0、12.5、25、50、100、200 μmol/L)NMN处理Huh7细胞24 h,采用细胞增殖及毒性检测试剂盒(CCK-8)检测NMN对细胞活力的影响；油红O染色观察细胞内脂质蓄积情况；免疫荧光显微镜观察细胞对DiI-LDL的摄取能力；实时定量PCR(qRT-PCR)和Western blot检测细胞内肝细胞核因子1α(HNF1α)、前蛋白转化酶枯草溶菌素9(PCSK9)、低密度脂蛋白受体(LDLR)的mRNA和蛋白表达情况。结果 CCK-8实验结果显示,不同浓度NMN处理Huh7细胞24 h对细胞活力没有显著影响；油红O染色显示,LDL加入后细胞内红色脂滴数量随着NMN浓度增加而增多；免疫荧光显微镜观察结果显示,细胞核周红色荧光随着NMN浓度增加而增强；Western blot和qRT-PCR结果显示,100、200 μmol/L NMN使Huh7细胞PCSK9、HNF1α mRNA和蛋白的表达显著降低,LDLR mRNA和蛋白的表达明显升高(P＜0.01)。结论 NMN可能通过介导HNF1α/PCSK9/LDLR信号通路参与调节Huh7细胞的胆固醇代谢,增强肝细胞对LDL的摄取能力。

    Abstract:

    Aim To explore the regulatory effect of nicotinamide mononucleotide (NMN) on cholesterol in Huh7 cells and its molecular mechanism. Methods Huh7 cells were treated with different concentrations of NMN (0,2.5,5, 0,0, 200 μmol/L) for 24 hours, and the cell viability was detected by cell counting kit-8 (CCK-8). The lipid accumulation in cells was observed by oil red O staining. The uptake ability of cells to DiI-LDL was observed by immunofluorescence microscope. Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expressions of mRNA and protein of intracellular hepatocyte nuclear factor 1α (HNF1α), proprotein convertase subtilisin kexin 9 (PCSK9), and low density lipoprotein receptor (LDLR). Results CCK-8 experiment showed that different concentrations of NMN had no significant effect on cell viability. Oil red O staining showed that the number of orange-red lipid droplets in the cells increased with the increase of NMN concentration after the addition of LDL. Immunofluorescence microscope observation results showed that the red fluorescence around the nucleus increased with the increase of NMN concentration. Western blot and qRT-PCR results showed that 100 and 200 μmol/L NMN significantly reduced the expressions of PCSK9 and HNF1α mRNA and protein, and significantly increased the expressions of LDLR mRNA and protein in Huh7 cells (P＜0.01). Conclusion NMN may be involved in regulating the cholesterol metabolism of Huh7 cells by mediating the HNF1α/PCSK9/LDLR signaling pathway, and enhance the uptake capacity of LDL by liver cells.

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引用本文

刘文凤,雷小灿,向琼,廖韦,夏梦蝶,彭娟.烟酰胺单核苷酸对Huh7细胞胆固醇代谢调节作用的研究[J].中国动脉硬化杂志,2021,29(8):668~674.

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  • 收稿日期:2021-04-23
  • 最后修改日期:2021-05-22
  • 在线发布日期: 2021-08-10

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