Hypomethylation of nerve growth factor (NGF) promotes binding of C/EBPα and contributes to inflammatory hyperalgesia in rats

Abstract

Background: Chronic pain usually accompanied by tissue damage and inflammation. However, the pathogenesis of chronic pain remains unclear.

Methods: We investigated the role of nerve growth factor (NGF) in chronic inflammatory pain induced by complete Freund's adjuvant (CFA), explored the methylation status of CpG islands in the promoter region of the NGF gene, and clarified the function and mechanism of C/EBPα-NGF signaling pathway from epigenetic perspective in the chronic inflammatory pain model.

Results: CFA induced significant hyperalgesia and continuous upregulation of NGF mRNA and protein levels in the L4-6 dorsal root ganglions (DRGs) in rats. Hypomethylation of CpG islands occurred in the NGF gene promoter region after CFA treatment. At the same time, the miR-29b expression level was significantly increased, while the DNA methyltransferase 3b (DNMT3b) level reduced significantly. Moreover, CFA treatment promoted binding of C/EBPα to the NGF gene promoter region and C/EBPα siRNA treatment obviously decreased expression of NGF levels and also alleviate inflammatory hyperalgesia significantly in rats.

Conclusion: Collectively, the results indicated that CFA leads to the upregulation of miR-29b level, which represses the expression of DNMT3b, enhances the demethylation of the NGF gene promoter region, and promotes the binding of C/EBPα with the NGF gene promoter, thus results in the upregulation of NGF gene expression and maintenance of chronic inflammatory pain.

Keywords: C/EBPα; DNA methylation; Epigenetics; Inflammatory pain; NGF.

Fig. 1

Dynamic changes of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) after CFA treatment. a 1 day after injection, the PWT of the CFA injection group (CFA) was significantly decreased (**P < 0.01, compared with the corresponding saline control group (Saline), n = 7). b The PWL was also significantly decreased in the CFA group compared to the corresponding saline-treated control group starting at 1 day after injection (**P < 0.01, n = 7)

Fig. 2

Upregulation of NGF expression in DRGs after CFA treatment. a, b NGF mRNA and protein expression were all increased significantly compared with the saline-treated group (CON) after CFA injection (*P < 0.05, **P < 0.01 compared with CON group, n = 4)

Fig. 3

Hypomethylation of CpG islands of the NGF gene promoter after CFA treatment. a Online software prediction of CpG islands around the NGF gene promoter region. The light blue areas on the map indicate the potential CpG islands. b The representative MSP assay showed DNA methylation and unmethylation ratio of the CpG islands in the NGF gene promoter region of L4–6 DRGs was significantly decreased after 3 days of CFA treatment (*P < 0.05, compared with the control group, n = 4). c Schematic of CpG island showing locations of the 12 CpG sites in the NGF gene promoter area d BSP sequencing, CFA injection resulted in a significant percentage of methylated CpG sites decrease to 16.7% in CpG islands of the NGF gene promoter region. e, f The expression of DNMT3b mRNA and protein in DRGs were significantly decreased after CFA treatment (3 days after CFA injection) (*P < 0.05, compared with the control group, n = 4), while DNMT3a level did not change significantly

Fig. 4

DNMT3b is a direct target of miR-29b in DRGs. a The expression of miR-29b was significantly increased in DRGs after CFA treatment (*P < 0.05, compared with the control group, n = 4). b The relative expression level of miR-29b when transfected with its inhibitor and mimic compared with scramble or miR-CON group, respectively (*P < 0.05, #P < 0.05). c,d Western blot analysis showed that overexpression of miR-29b inhibited the protein level of DNMT3b, and inhibition of miR-29b expression promoted the expression of DNMT3b protein (*P < 0.05, #P < 0.05). e The reverse binding sequence of mature miR-29b inserted into C-terminal of the luciferase gene to generate pGL3-DNMT3b -3’UTR. f miR-29b targeted the wild type but not the mutant 3’UTR of DNMT3b (*P < 0.05)

Fig. 5

Relative C/EBPα expression levels are upregulated significantly in DRGs after CFA treatment. a, b Relative C/EBPα mRNA and protein expressions were all increased significantly compared with the saline-treated group (CON) after 1 day of CFA injection (*P < 0.05, **P < 0.01 compared with CON group, n = 4)

Fig. 6

Promoted binding of C/EBPα to the NGF gene promoter region in DRGs after CFA treatment. a Two predicted C/EBPα binding sites in the CpG islands of the NGF gene promoter. b, c Chromatin immunoprecipitation assays indicate a significant increase in binding activity of C/EBPα with the two sites of NGF gene promoter in CFA-induced inflammatory rats when compared with control rats (*P < 0.05, compared with the control group, n = 4 for each group)

Fig. 7

C/EBPα siRNA downregulates the expression level of NGF. a C/EBPα siRNA treatment obviously reverses the expression of NGF levels when 3 days after CFA treatment (*P < 0.05, compared with NC siRNA group, n = 4 for each group). b, c C/EBPα siRNA significantly alleviate inflammatory hyperalgesia in rats that induced by CFA (*P < 0.05, **P < 0.01, compared with NC group, n = 4 for each group)

Fig. 8

Schematic shows the regulation of the NGF gene expression and the involvement of C/EBPα in chronic inflammatory pain. Chronic inflammation leads to upregulation of miR-29b expression level which represses the activity of DNMT3b, enhances the demethylation of the NGF gene promoter region, and promotes the binding of C/EBPα with NGF gene promoter, resulting in upregulation of NGF gene expression, and contribute to the maintenance of chronic inflammatory pain