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

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.

Abstract

We found Dinucleotide methylation status of CpG islands in the IL‐6 promotor region correlated ith IL‐6 expression in murine macrophages. Alpinetin could induce dinucleotide methylation status of CpG islands in the IL‐6 promotor region by activating methyltransferase, thus inhibiting IL‐6 expression.