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

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, 5, 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)(n=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 decline 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 (P < 0.01), while were decreased in the MH group than the NIC group (P < 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 (P < 0.05, P < 0.01), while those of related molecules were significantly reduced in the MH group than the NIC group (P < 0.05, P < 0.01). The protein and mRNA levels of PCSK9 were upregulated in the NIC group (P < 0.05), while those in the MH, PLX, PD and CC groups were decreased than the NIC group (P < 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.

Keywords

• nicotine
• pcsk9
• ldl-c
• lipid metabolism
• hepg2 cells