Upregulation of CXCR4 through promoter demethylation contributes to inflammatory hyperalgesia in rats

Abstract

Aim and methods: Chronic pain associated with inflammation is a common clinical problem, and the underlying mechanisms yet are incompletely defined. DNA methylation has been implicated in the pathogenesis of chronic pain. However, the specific genes regulated by DNA methylation under inflammatory pain condition remain largely unknown. Here, we investigated how chemokine receptor CXCR4 expression is regulated by DNA methylation and how it contributes to inflammatory pain induced by complete Freund's adjuvant (CFA) in rats.

Results: Intraplantar injection of CFA could not only induce significant hyperalgesia in rats, but also significantly increase the expression of CXCR4 mRNA and protein in the dorsal root ganglion (DRG). Intrathecal injection of CXCR4 antagonist AMD3100 significantly relieved hyperalgesia in inflammatory rats in a time- and dose-dependent manner. Bisulfite sequencing and methylation-specific PCR demonstrate that CFA injection led to a significant demethylation of CpG island at CXCR4 gene promoter. Consistently, the expression of DNMT3b was significantly downregulated after CFA injection. Online software prediction reveals three binding sites of p65 in the CpG island of CXCR4 gene promoter, which has confirmed by the chromatin immunoprecipitation assay, CFA treatment significantly increases the recruitment of p65 to CXCR4 gene promoter. Inhibition of NF-kB signaling using p65 inhibitor pyrrolidine dithiocarbamate significantly prevented the increases of the CXCR4 expression.

Conclusion: Upregulation of CXCR4 expression due to promoter demethylation followed by increased recruitment of p65 to promoter of CXCR4 gene contributes to inflammatory hyperalgesia. These findings provide a theoretical basis for the treatment of chronic pain from an epigenetic perspective.

Keywords: CXCR4; DNA methylation; dorsal root ganglion; epigenetics; inflammatory pain.

Figure 1

Dynamic changes of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) after injection of CFA. (A) After 12 h of CFA injection, the rats in the model group present significant mechanical hyperalgesia, and the PWT was significantly reduced (**P < 0.01 compared with the corresponding saline control group (Saline), n = 7). (B) After injection of CFA for 12 h, the PWL was significantly decreased in the model group compared with the corresponding saline‐treated control group (*P < 0.05, **P < 0.01, n = 7)

Figure 2

Coexpression of CXCR4 with β‐tubulin III in the hind paw's plantar projecting DRG neurons. CXCR4‐positive cells are shown in green (top left), β‐tubulin III‐positive cells are shown in blue (top middle), the hind paw's plantar surface projecting DRG cells were labeled with DiI (red, top right) injected into the plantar surface of hind paw. Merge of β‐tubulin III‐positive staining and CXCR4 labeling (bottom left). Merge of double labeling of DiI and CXCR4 (bottom middle). Merge of β‐tubulin III‐positive staining and DiI labeling (bottom right). Scale bar, 50 μm

Figure 3

The changes of CXCR4 expression after injection of CFA. The expression of CXCR4 mRNA and protein in the L4‐6 DRGs of the inflamed rats was significantly increased. Compared with the saline‐treated group (CON), CXCR4 mRNA (A) and protein (B) expression were significantly increased after 12 h of CFA injection (*P < 0.05, **P < 0.01 compared with CON group, n = 4)

Figure 4

Inflammatory pain can be significantly relieved by CXCR4 inhibitor AMD3100 in rats. (A) The effects of different doses of AMD3100 (5, 25 and 50 μg/kg) on the mechanical pain in rats were observed. The optimal dose of 25 μg/kg could significantly relieve inflammatory pain in rats, which can be sustained for 8 hours (*P < 0.05,**P < 0.01 compared to the corresponding saline group, n = 7). (B) The effect of different doses of AMD3100 (5, 25 and 50 μg/kg) on the thermal pain in rats was observed. The optimal dose of 25 μg/kg could significantly reverse the pain behavior of rats, which can be sustained for 8 hours (*P < 0.05,**P < 0.01 compared with the corresponding saline group, n = 7)

Figure 5

MSP results showed DNA methylation of CpG islands in the CXCR4 gene promoter region of L4‐6 DRGs. (A) Online software prediction of CpG island around the CXCR4 gene promoter region. The light blue areas on the map indicate the potential CpG islands. (B) The MSP results showed that the DNA methylation and demethylation ratio of the CpG island in the CXCR4 gene promoter region of L4‐6 DRGs was significantly decreased after 3 days of CFA injection (*P < 0.05, compared with the control group, n = 4)

Figure 6

BSP results showed DNA methylation of CpG islands in the CXCR4 gene promoter region of L4‐6 DRGs. (A) CpG island position diagram of CXCR4 gene promoter region, and BSP DNA sequencing sequence. (B) BSP sequencing, CFA injection resulted in a significant decrease in CpG island methylation in the CXCR4 gene promoter region (*P < 0.05, compared with the control group, n = 4). (C) Expression levels of DNMT3a and 3b in L4 ‐6 DRGs by qPCR. The expression of DNA methylase DNMT3b in DRGs was significantly decreased after CFA induction (3 d after CFA injection) (*P < 0.05, compared with the control group, n = 4), while DNMT3a level did not change significantly

Figure 7

Enriched recruitment of p65 to CXCR 4 gene promoter in DRGs of CFA‐induced inflammatory rats. (A‐C) Chromatin immunoprecipitation assays indicated a significant increase in binding activity of p65 with the second and third binding sites of promoter of CXCR4 gene in CFA‐induced inflammatory rats compared with age‐matched CON (*P < 0.05, compared with the control group, n = 4 for each group). However, there is no significant change in binding activity of p65 with the first binding site of promoter of CXCR4 gene in CFA‐induced inflammatory rats compared with age‐matched CON rats (n = 4 for each group). (D) Reversed upregulation of CXCR4 expression by PDTC. Intraperitoneal injection of NF‐kB inhibitor PDTC once daily for consecutive 3 days markedly reversed the upregulation of CXCR4 in CFA‐induced inflammatory rats (*P < 0.05, **P < 0.01 compared to the corresponding saline‐treated group, n = 5)