Inhibitory effect of alpinetin on IL-6 expression by promoting cytosine methylation in CpG islands in the IL-6 promoter region

Ke Hu; Yuxian Li; Minghua Liang; Lijing Liu; Yuefu Chen; Minjiang Huang; Bifeng Tan; Yingquan Luo; Huiming Yin

doi:10.1002/mgg3.993

Inhibitory effect of alpinetin on IL-6 expression by promoting cytosine methylation in CpG islands in the IL-6 promoter region

Mol Genet Genomic Med. 2020 Jan;8(1):e993. doi: 10.1002/mgg3.993. Epub 2019 Nov 13.

Authors

Ke Hu¹, Yuxian Li¹, Minghua Liang², Lijing Liu¹, Yuefu Chen¹, Minjiang Huang¹, Bifeng Tan³, Yingquan Luo⁴, Huiming Yin⁵

Affiliations

¹ Medical college, Hunan University of Medicine, Huaihua, Hunan, PR China.
² Department of Pediatrics, first people's hospital of huaihua, Huaihua, Hunan, PR China.
³ Department of Cardiology, first affiliated hospital, Hunan University of Medicine, Huaihua, Hunan, PR China.
⁴ Department of Senile Disease, second Xiangya hospital, Central South University, Changsha, Hunan, PR China.
⁵ Department of Respiration, first affiliated hospital, Hunan University of Medicine, Huaihua, Hunan, PR China.

Abstract

Background: Alpinetin is a flavonoid which exerts antibacterial and anti-inflammatory functions. In order to prove that the induced methylation is an important mechanism for alpinetin in regulating the expression of inflammatory factor Interleukin-6 (IL-6), we detected the dinucleotide methylation status of CpG islands in the IL-6 promoter region and IL-6 level after treatment of RAW246.7 murine macrophages with alpinetin.

Methods: After RAW246.7 murine macrophages were treated with alpinetin, alpinetin + GW9662 (the peroxisome proliferator-activated receptor (PPAR) antagonist), and alpinetin + DNA methyltransferase 3 alpha (DNMT3A) siRNA for 96 hr, CpG islands were analyzed using time-of-flight mass spectrophotometry (TOF-MS) and bisulfite sequencing polymerase chain reaction (BSP). Dinucleotide methylation status of the CpG islands in the IL-6 promoter region was analyzed by methylation-specific Polymerase Chain Reaction (PCR). IL-6 level was detected using the enzyme-linked immunosorbent assay (ELISA) method. Pearson's correlation analysis was conducted to test for potential correlation between the methylation status of CpG islands in the IL-6 promoter region and IL-6 level in RAW 246.7 cells.

Results: Alpinetin promoted dinucleotide methylation status of two CpG islands in the IL-6 promoter region stretching 500-2500 bp upstream of the transcriptional start site (TSS) (p < .05). This promoting effect was more significant for the CpG island stretching 500-1500 bp long. The methylation ratio of dinucleotide at this position was significantly inversely correlated with the level of IL-6 (p < .05). PPAR antagonist GW9662 and interference of DNMT3A could reverse both the alpinetin-induced methylation and inhibitory effects on IL-6 expression.

Conclusion: Alpinetin could induce dinucleotide methylation status of CpG islands in the IL-6 promoter region by activating methyltransferase, thus inhibiting IL-6 expression in murine macrophages.

Keywords: Alpinetin; CpG island; IL-6; methylation; murine macrophage; promoter region.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

5-Methylcytosine / metabolism
Anilides / pharmacology
Animals
Anti-Inflammatory Agents / pharmacology*
CpG Islands
DNA (Cytosine-5-)-Methyltransferases / genetics
DNA (Cytosine-5-)-Methyltransferases / metabolism
DNA Methylation / drug effects*
DNA Methyltransferase 3A
Flavanones / pharmacology*
Interleukin-6 / genetics*
Interleukin-6 / metabolism
Mice
Peroxisome Proliferator-Activated Receptors / antagonists & inhibitors
Promoter Regions, Genetic
RAW 264.7 Cells

Substances

2-chloro-5-nitrobenzanilide
Anilides
Anti-Inflammatory Agents
Dnmt3a protein, mouse
Flavanones
Interleukin-6
Peroxisome Proliferator-Activated Receptors
alpinetin
interleukin-6, mouse
5-Methylcytosine
DNA (Cytosine-5-)-Methyltransferases
DNA Methyltransferase 3A