Abstract
BackgroundAlpinetin 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.MethodsAfter 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.ResultsAlpinetin 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.ConclusionAlpinetin 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.
Highlights
Inflammatory damage is considered as the main threat to human health, which is intended to restore the steady‐state level of inflammatory factors (Abdelhalim, Moussa, Qaid, & Al‐Ayed, 2018)
Further understanding on the anti‐inflammatory effects and mechanism of Researchers have already conducted a full research into the anti‐inflammatory mechanism of alpinetin and found that the nuclear factor kappa B (NF‐кBs) and extracellular regulates protein kinases (ERKs) signaling pathways are inhibited after alpinetin activates PPAR, leading to a reduced synthesis of inflammatory factors (Liu et al, 2017; Ma et al, 2017)
We detected intranuclear deacetylase activity in RAW246.7 cells following alpinetin treatment and proved that the increased activity of DNA methyltransferase 3 alpha (DNMT3A) is related to the deacetylation of CpG binding to the promoter region of the inflammatory factors
Summary
Inflammatory damage is considered as the main threat to human health, which is intended to restore the steady‐state level of inflammatory factors (Abdelhalim, Moussa, Qaid, & Al‐Ayed, 2018). The pharmacological effect of flavonoids is related to the activation of PPARs which inhibits the expression of inflammatory mediators through several pathways. Previous study indicated that alpinetin inhibits the expressions of intracellular inflammatory signaling pathways after activating PPARs, while inhibit the synthesis of upstream transcriptional factors of inflammatory genes such as tumor necrosis factor α (TNF‐α), IL‐1ß, and IL‐6. This study indicated that alpinetin may regulate the expressions of the target genes by inducing methylation after activating PPAR (Liang et al, 2016). We detected the effects and mechanisms of alpinetin on dinucleotide methylation status of CpG islands in the IL‐6 promoter region and IL‐6 level in RAW246.7 murine macrophages, for providing the basis for the clinical use of alpinetin
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