Ligand-gated ion channel P2X7 regulates hypoxia-induced factor-1α mediated pain induced by dental pulpitis in the medullary dorsal horn

Abstract

Dental pulpitis often induces severe pain, and the molecular immune response is remarkable in both peripheral and central nervous system. Accumulating evidence indicates that activated microglia in the medullary dorsal horn (MDH) contribute to dental pulpitis induced pain. The P2X7 receptor plays an important role in driving pain and inflammatory processes, and its downstream target hypoxia-induced factor-1α (HIF-1α) has a crucial role in maintaining inflammation. However, the relationship between P2X7 and HIF-1α in dental inflammatory pain remains unclear. This study demonstrated that the degree of inflammation in the dental pulp tissue became more severe in a time-dependent manner by establishing a rat dental pulpitis model via pulp exposure. Meanwhile, the expression of P2X7, HIF-1α, IL-1β, and IL-18 in the MDH increased most on the seventh day when the pain threshold was the lowest in the dental pulpitis model. Furthermore, lipopolysaccharides (LPS) increased P2X7-mediated HIF-1α expression in microglia. Notably, the suppression of P2X7 caused less IL-1β and IL-18 release and lower HIF-1α expression, and P2X7 antagonist Brilliant Blue G (BBG) could alleviate pain behaviors of the dental pulpitis rats. In conclusion, our results provide further evidence that P2X7 is a key molecule, which regulates HIF-1α expression and inflammation in dental pulpitis-induced pain.

Keywords: HIF-1α; P2X7; dental pulpitis; medullary dorsal horn; microglia; pain.

Figure 1

Establishment of the rat dental pulpitis pain model. (A) Schedule of pulp exposure treatment and behavioral test. (B) A rat dental pulpitis model was established via dental pulp exposure. (C) The pain threshold decreased with a prolonged exposure time of the dental pulp, reaching the minimum on the seventh day, and then increasing slightly during the next 7 days. *p < 0.05 vs. control. (D) Haematoxylin and eosin (H&E) staining showed that the inflammatory infiltration range in dental pulpitis tissue gradually expanded from the crown to the root pulp as the exposure time prolonged. Scale bar = 100 μm.

Figure 2

Activation of microglia and release of inflammatory factors in medullary dorsal horn (MDH) after dental pulp exposure. (A) The location of the MDH in the representative coronal brain slices schematic diagram. (B) The expression of Iba-1 after dental pulp exposure. (C) The protein levels of IL-1β and IL-18 in MDH. (D) Pearson correlation analysis of the withdrawal threshold and IL-1β and IL-18 protein levels in MDH. *p < 0.05, **p < 0.01 vs. 7 d group, ^***p < 0.001 vs. 7 days group, ^##p < 0.01, ^###p < 0.001 vs. control.

Figure 3

Expression of P2X7 in MDH after dental pulp exposure. (A,B) Immunofluorescence staining showed that expression of P2X7 in MDH gradually increased with a prolonged exposure time of the dental pulp. (C) Western blot analysis showed that P2X7 protein levels markedly increased compared to the control group, with the highest expression on the seventh day. (D) The mRNA levels of P2X7 increased gradually with a prolonged pulp exposure time with the highest expression on the seventh day. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. 7 days group, ^##p < 0.01, ^###p < 0.001 vs. control. (E) Double immunofluorescence staining showed that P2X7 was mainly located in microglia, rather than in astrocytes nor neurons. ^##p < 0.01, ^###p < 0.001 vs. Iba-1 group.

Figure 4

Expression of HIF-1α in MDH after dental pulp exposure. (A,B) Immunofluorescence staining showed that expression of HIF-1α in MDH gradually increased with a prolonged exposure time of the dental pulp. (C) Western blot analysis showed that HIF-1α protein levels significantly increased compared to the control group, with the highest expression on the seventh day. (D) The mRNA levels of HIF-1α increased with the highest expression on the seventh day. (E) Double immunofluorescence staining revealed that P2X7 is colocalized with HIF-1α in MDH. ^***p < 0.001 vs. 7 days group, ^#p < 0.05, ^##p < 0 0.01, and ^###p < 0.001 vs. control.

Figure 5

Effects of P2X7 on HIF-1α expression and inflammatory factors levels in BV2 cells. (A) Western blot results showed that LPS-induced upregulation of HIF-1α expression which was inhibited by oxATP. ^###p < 0.001 vs. control, ***p < 0.001 vs. LPS group. (B) HIF-1α expression was downregulated with P2X7 siRNA pretreatment. ^***p < 0.001 vs. negative control group. (C) LPS induced upregulation of IL-1β and IL-18. ^***p < 0.001 vs. control. (D) The protein expression levels of IL-1β and IL-18 in BV2 cells treated with siRNA against P2X7. ^***p < 0.001 vs. negative control group.

Figure 6

Effects of Brilliant Blue G (BBG) on pain behaviors and the expression of P2X7, HIF-1α, and inflammatory factors. (A) The pain threshold of the sham, vehicle and BBG rats. ^*p < 0.05 vs. sham, ^#p < 0.05 vs. vehicle. (B) The effect of dental pulp exposure and BBG treatment on mRNA level of P2X7 in MDH. *p < 0.05 vs. sham, ^##p < 0.01 vs. vehicle. (C) The effect of dental pulp exposure and BBG treatment on mRNA level of HIF-1α in MDH. ***p < 0.001 vs. sham, ^###p < 0.01 vs. vehicle. (D) The effect of dental pulp exposure and BBG treatment on mRNA level of IL-1β in MDH. ^***p < 0.01 vs. sham, ^##p < 0.01 vs. vehicle. (E) The effect of dental pulp exposure and BBG treatment on mRNA level of IL-18 in MDH. ^***p < 0.001 vs. sham, ^###p < 0.01 vs. vehicle.