Combination of resolvin E1 and lipoxin A4 promotes the resolution of pulpitis by inhibiting NF-κB activation through upregulating sirtuin 7 in dental pulp fibroblasts

Abstract

Objectives: To determine whether the combination of resolvin E1 (RvE1) and lipoxin A4 (LXA4) could promote resolution of pulpitis and to investigate the mechanism.

Materials and methods: Preliminary screening was first conducted in four specialized pro-resolving mediators (SPMs). Real-time quantitative polymerase chain reaction, western blotting, enzyme-linked immunosorbent assay and double-immunofluorescence labelling were employed to assess the expression of RelA, SIRT1, SIRT6, SIRT7 and pro-inflammatory factors. Dental pulp fibroblasts (DPFs) were transfected with siRNA to assess the biological role of SIRT7. A pulpitis model was utilized to evaluate the in vivo curative effect.

Results: Preliminary results showed that RvE1 and LXA4 reduced the expression of RelA more markedly than other two SPMs. Both RvE1 and LXA4 treatment downregulated nuclear factor kappa B (NF-κB) activation and increased the expression of SIRT1, SIRT6 and SIRT7, more so in combination than alone. Double-immunofluorescence labelling showed that SIRT7 co-localized with p-p65 and Ac-p65 in the nucleus. Inhibiting ChemR23 and ALX reversed the expression of RelA mRNA, p-p65 and Ac-p65 proteins, pro-inflammatory factors, SIRT1, SIRT6 and SIRT7. Silencing SIRT7 significantly increased p-p65 and Ac-p65 protein levels and decreased SIRT1 and SIRT6 expression. In vivo experiments showed that combined administration of RvE1 and LXA4 promoted pulpitis markedly to resolution.

Conclusions: Combination of RvE1 and LXA4 effectively inhibited NF-κB activation by upregulating SIRT7 expression in DPFs, leading to reduced production of pro-inflammatory factors and promotion of pulpitis resolution.

FIGURE 1

Effects of combined administration of RvE1 and LXA4 on pro‐inflammatory factor expression. (A) Expression of RelA mRNA in LPS‐induced DPFs after treatment with four representative specialized pro‐resolving mediators. (B) Expression of RelA mRNA in LPS‐induced DPFs after treatment with RvE1 or LXA4 alone, and a combination of RvE1 and LXA4. (C) NLRP3, (D) caspase‐1, (E) IL‐1β and (F) IL‐18 mRNA levels on LPS‐induced DPFs detected by qPCR and (G) their protein levels tested by western blotting (normalized to that of β‐tubulin). (H) IL‐1β and (I) IL‐18 protein levels from cultural supernatant detected by ELISA. Other NF‐κB‐dependent expression of genes (J) IL‐6, (K) TNF‐α, (L) CCL2 and (M) CCL7 detected by qPCR. (*p < 0.05 and **p < 0.01). DPFs, dental pulp fibroblasts; ELISA, enzyme‐linked immunosorbent assay; LPS, lipopolysaccharide; LXA4, lipoxin A4; NF‐κB, nuclear factor kappa B; qPCR, quantitative polymerase chain reaction; RvE1, resolvin E1

FIGURE 2

Effects of the RvE1 and LXA4 combination on NF‐κB modification. (A) NAD+/NADH ratio measured by a NAD+/NADH assay kit. (B) SIRT1, (C) SIRT6, and (D) SIRT7 mRNA levels detected by qPCR. (E) Expression of p65 protein in both cytoplasm and nucleus detected by western blotting, normalized to β‐actin (in cytoplasm) or PCNA (in nucleus). (F) Phosphorylation and acetylation levels of p65 and expressions of SIRT1, SIRT6 and SIRT7 protein (normalized to that of β‐tubulin) detected by western blotting. (G) Representative double‐immunofluorescence labelling images of SIRT7 (green) and p‐p65 (red), and the nuclei were stained with DAPI (blue). (H) Representative double‐immunofluorescence labelling images of SIRT7 (green) and Ac‐p65 (red), and the nuclei were stained with DAPI (blue). (*p < 0.05 and **p < 0.01). DAPI, 4′,6‐diamidino‐2‐phenylindole; LXA4, lipoxin A4; NAD+, nicotinamide adenine dinucleotide; NF‐κB, nuclear factor kappa B; qPCR, quantitative polymerase chain reaction; RvE1, resolvin E1

FIGURE 3

Pro‐resolving effect of the RvE1 and LXA4 combination after ChemR23 and/or ALX inhibition. (A) Expression of RelA mRNA detected by qPCR. (B) NAD+/NADH ratio measured by an NAD+/NADH assay kit. (C) SIRT1, (D) SIRT6 and (E) SIRT7 mRNA levels detected by qPCR. (F) Expression of p65 protein in both cytoplasm and nucleus detected by western blotting, normalized to β‐actin (in cytoplasm) or PCNA (in nucleus). (G) Phosphorylation and acetylation levels of p65 and expressions of SIRT1, SIRT6 and SIRT7 protein (normalized to β‐tubulin) detected by western blotting. (H) Representative double‐immunofluorescence labelling images of SIRT7 (green) and p‐p65 (red), and the nuclei were stained with DAPI (blue). (I) Representative double‐immunofluorescence labelling images of SIRT7 (green) and Ac‐p65 (red), and the nuclei were stained with DAPI (blue). (*p < 0.05 and **p < 0.01). DAPI, 4′,6‐diamidino‐2‐phenylindole; LXA4, lipoxin A4; NAD+, nicotinamide adenine dinucleotide; qPCR, quantitative polymerase chain reaction; RvE1, resolvin E1

FIGURE 4

Pro‐resolving effect of the RvE1 and LXA4 combination after SIRT7 silencing. (A) Silencing efficiency of si‐SIRT7 on mRNA level, and (B) that of in protein level. (C) Expression of RelA mRNA detected by qPCR. (D) NAD+/NADH ratio measured by an NAD+/NADH assay kit. (E) SIRT1 and (F) SIRT6 mRNA levels detected by qPCR. (G) Expression of p65 protein in both cytoplasm and nucleus detected by western blotting, normalized to β‐actin (in cytoplasm) or PCNA (in nucleus). (H) Phosphorylation and acetylation levels of p65 and expressions of SIRT1, SIRT6 and SIRT7 protein (normalized to β‐tubulin) detected by western blotting. (I) Representative double‐immunofluorescence labelling images of p‐p65 (green) and Ac‐p65 (red), and the nuclei were stained with DAPI (blue). (*p < 0.05 and **p < 0.01). DAPI, 4′,6‐diamidino‐2‐phenylindole; LXA4, lipoxin A4; NAD+, nicotinamide adenine dinucleotide; qPCR, quantitative polymerase chain reaction; RvE1, resolvin E1

FIGURE 5

Therapeutic effect of combined administration of RvE1 and LXA4 in a rat pulpitis model. (A) Representative haematoxylin–eosin‐stained images on days 1, 3 and 7 after surgery. (B) Representative immunofluorescence images of CD11b on days 1, 3 and 7 after surgery. (C) Representative immunohistochemistry‐stained images of OCN on days 3 and 7 after surgery. (D) Statistical analysis of number of immune cells. (E) Statistical analysis of the area of mineralized tissue. (*p < 0.05 and **p < 0.01). LXA4, lipoxin A4; RvE1, resolvin E1

FIGURE 6

(A) Representative immunohistochemistry‐stained images of NLRP3, caspase‐1, IL‐1β, and IL‐18 of day 3 samples. (B) Isotype control. Statistical analysis of (C) NLRP3‐, (D) caspase‐1‐, (E) IL‐1β‐ and (F) IL‐18‐positive cells. (*p < 0.05 and **p < 0.01)

FIGURE 7

The molecular mechanism by which the RvE1 and LXA4 combination resolves pulpitis as proposed in the present study. LXA4, lipoxin A4; RvE1, resolvin E1