Nicotine regulates PCSK9 expression in HepG2 cells through Raf/MEK/ERK signaling pathway

Lin ZHOU; Rui XIANG; Min MAO; Rui FENG; Yuhao HU; Yueming CHEN; Jie CHEN; Qiuyue SHI; Qin LOU; Jing CHANG

doi:10.16016/j.2097-0927.202301028

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Nicotine regulates PCSK9 expression in HepG2 cells through Raf/MEK/ERK signaling pathway

Basic Medicine | 更新时间：2026-01-09

- Nicotine regulates PCSK9 expression in HepG2 cells through Raf/MEK/ERK signaling pathway
- Journal of Army Medical University Vol. 45, Issue 14, Pages: 1547-1555(2023)
- 作者机构：
  
  400016 重庆，重庆医科大学附属第一医院心血管内科，重庆市重大代谢性疾病转化医学重点实验室
- 作者简介：
- 基金信息：
  
  the Natural Science Foundation of Chongqing(CSTC2020jcyj-msxmX0853)
- DOI：10.16016/j.2097-0927.202301028
  CLC： Q591.5;R345;R996
- Received：07 January 2023，
  
  Revised：2023-04-07，
  
  Published：30 July 2023
- 稿件说明：
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Lin ZHOU, Rui XIANG, Min MAO, et al. Nicotine regulates PCSK9 expression in HepG2 cells through Raf/MEK/ERK signaling pathway[J]. Journal of Army Medical University, 2023, 45(14): 1547-1555.
DOI：

Lin ZHOU, Rui XIANG, Min MAO, et al. Nicotine regulates PCSK9 expression in HepG2 cells through Raf/MEK/ERK signaling pathway[J]. Journal of Army Medical University, 2023, 45(14): 1547-1555. DOI： 10.16016/j.2097-0927.202301028.

摘要

目的

探讨尼古丁对前蛋白转化酶枯草杆菌蛋白酶/kexin 9型(proprotein convertase subtilisin/kexin type 9

PCSK9)表达的影响以及分子机制。

方法

采用不同浓度的尼古丁(0、5、10、20 μmol/L)处理HepG2细胞0、24、48 h，观察与低密度脂蛋白胆固醇(low density lipoprotein cholesterol

LDL-C)代谢密切相关的PCSK9的变化情况。实验分(

=3)：对照组(CON组，细胞正常培养)；尼古丁组(NIC组，加入10 μmol/L的尼古丁孵育24 h)；尼古丁+尼古丁抑制剂组(MH组，10 μmol/L尼古丁抑制剂盐酸美加明)；尼古丁+Raf抑制剂组[PLX组，1 μmol/L Raf抑制剂(PLX4720)

]

；尼古丁+MEK抑制剂组[PD组，1 μmol/L MEK抑制剂(PD0325901)

]

；尼古丁+ERK抑制剂组[CC组，1 μmol/L ERK抑制剂(CC-90003)

]

。后4组预处理1 h后再加入10 μmol/L的尼古丁一起孵育24 h。采用CCK-8实验检测HepG2细胞的活性，划痕实验检测细胞迁移能力，油红O染色检测尼古丁处理后的脂质沉积。运用网络药理学筛选LDL-C与尼古丁的共同靶点和作用通路。分别使用Western blot和RT-qPCR分析PCSK9以及MAPK通路(Raf、MEK、ERK)的蛋白和mRNA的表达水平。用免疫荧光染色、酶联免疫吸附实验(ELISA)检测PCSK9蛋白的表达水平。

结果

随着尼古丁浓度的升高，HepG2细胞的活性不断降低，并且相同尼古丁浓度下48 h比24 h下降更多。与CON组比较，NIC组HepG2细胞的迁移能力和脂质沉积增加(

0.01)，而MH组相较于NIC组迁移能力和脂质沉积下降(

0.01)。LDL-C与尼古丁的交集靶点多达257个，二者的蛋白质-蛋白质相互作用(protein-protein interaction networks，PPI)核心靶点涉及MAPK通路；生物过程包含“MAPK活性的正向调节”，且通路分析中的“脂质和动脉粥样硬化”富集分数最高。与CON组比较，NIC组Raf、MEK、ERK的蛋白和mRNA表达增强(

0.05，

0.01)，MH组的相关蛋白和mRNA表达较NIC组明显下调(

0.05，

0.01)。NIC组的PCSK9蛋白和mRNA较CON组上调(

0.05)，MH组、PLX组、PD组和CC组的PCSK9蛋白和mRNA较NIC组下降(

0.05)。

结论

尼古丁可增加HepG2细胞脂质沉积和迁移能力，并通过Raf/MEK/ERK信号通路调节HepG2细胞中PCSK9的表达；上调的PCSK9可能是细胞内脂质沉积的重要原因。

Abstract

Objective

To investigate the effect of nicotine (NIC) on the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) and its molecular mechanism.

Methods

Different concentrations of NIC (0

10 and 20 μmol/L) were used to treat HepG2 cells for 0

24 or 48 h to observe the change of PCSK9

which is closely related to the metabolism of low-density lipoprotein cholesterol (LDL-C). Then the cells were grouped into control group (normal culture medium)

NIC group (10 μmol/L for 24 h)

NIC+inhibitor (MH) group (10 μmol/L NIC inhibitor

mecamylamine hydrochloride)

NIC+Raf inhibitor (PLX) group (1 μmol/L Raf inhibitor

PLX4720)

NIC+MEK inhibitor (PD) group (1 μmol/L MEK inhibitor

PD0325901)

and NIC+ERK inhibitor (CC) group (1 μmol/L ERK inhibitor

CC-90003)(

=3). The cells from the latter 4 groups were pretreated with corresponding agents for 1 h followed by 10 μmol/L NIC co-culture for 24 h. CCK-8 assay

cell scratch test and oil red O staining were used to detect cell viability

migration and lipid deposition

respectively. Then network pharmacology was employed to screen the common targets and action pathways of LDL-C and NIC. Western blotting and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) were performed to measure the protein and mRNA levels of PCSK9 and Raf

MEK and ERK in MAPK pathway. The expression level of PCSK9 was directly detected by immunofluorescence staining and ELISA.

Results

As the concentration of NIC increased

viability of HepG2 cells was decreased continuously

with the dec

line more significant at 48 h than at 24 h. The migration ability and lipid deposition of HepG2 cells were increased in the NIC group than the CON group (

0.01)

while were decreased in the MH group than the NIC group (

0.01). There are 257 intersection targets of LDL-C and NIC

and the core target of their protein-protein interaction (PPI) involves MAPK pathway. Biological process included "positive regulation of MAPK activity"

and the enrichment fraction of "lipid and atherosclerosis" got the highest in pathway analysis. Compared with the CON group

the protein and mRNA levels of Raf

MEK and ERK were increased in the NIC group (

0.05

0.01)

while those of related molecules were significantly reduced in the MH group than the NIC group (

0.05

0.01). The protein and mRNA levels of PCSK9 were upregulated in the NIC group (

0.05)

while those in the MH

PLX

PD and CC groups were decreased than the NIC group (

0.05).

Conclusion

NIC enhanced lipid deposition and migration ability and regulates the expression of PCSK9 through the Raf/MEK/ERK signaling pathway in HepG2 cells. And the up-regulation of PCSK9 may be an important cause of intracellular lipid deposition.

关键词

Keywords

references

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