Progranulin (PGRN) Facilitates Anti‐Inflammation and Pulpitis Repair In Vivo and In Vitro Through TNFR2/14‐3‐3ε Signalling Complex

AIM: To investigate the role and mechanism of progranulin (PGRN) in reparative dentinogenesis and inflammation control for rat pulpitis and inflammatory human dental pulp stem cells (hDPSCs). METHODOLOGY: Eight-week-old male Wistar rats with irreversible pulpitis were treated with pulpotomy and divided into five groups: No treatment; Control; iRoot BP plus (BP); GelMA and recombinant human PGRN (rhPGRN) + GelMA (rhPGRN). Micro-computed tomography (Micro-CT) scans and histological and immunohistochemical staining were conducted to evaluate rhPGRN' anti-inflammation and pro-healing properties. The effects of rhPGRN on hDPSC inflammatory response, proliferation and dentinogenic differentiation and potential signalling pathways were assessed through CCK-8, alkaline phosphatase (ALP) staining, alizarin red staining, quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining and western blotting. RESULTS: In vivo, PGRN expression obviously increased in both the Control and GelMA groups compared to healthy pulp (p < 0.05). The BP and rhPGRN groups showed a significant decrease in inflammatory scores and expression of M1 macrophage markers CD86 and tumour necrosis factor alpha (TNF-α) while increasing M2 markers CD206 and interleukin 10 (IL-10) compared with the controls (p < 0.05). Enhanced dentine bridge formation and dentine sialophosphoprotein (DSPP) expression were observed in the BP and rhPGRN groups versus the controls (p < 0.05). Moreover, the rhPGRN group presented higher expressions of CD206, IL-10 and DSPP than the BP group (p < 0.05). In vitro, PGRN expression significantly increased in lipopolysaccharide (LPS)-stimulated hDPSCs (p < 0.05). rhPGRN significantly reduced the release of TNF-α, interleukin 1 beta (IL-1β) and IL-6 in LPS-stimulated hDPSCs and enhanced ALP activity, mineralized nodule formation and expression of ALP, Runt-related transcription factor 2 (RUNX2) and DSPP in LPS-stimulated or unstimulated hDPSCs (p < 0.05). Mechanistically, co-immunoprecipitation showed that PGRN bound to tumour necrosis factor receptor-2 (TNFR2), interacting with 14-3-3 epsilon (14-3-3ε) in hDPSCs. PGRN significantly inhibited LPS-activated phosphorylation of NF-κB/p65 and its nuclear translocation, and the use of a TNFR2 neutralising antibody or the 14-3-3 protein inhibitor R18 reversed these effects (p < 0.05). CONCLUSION: These findings suggest that PGRN plays a crucial role in anti-inflammation, immunomodulation and reparative dentinogenesis in rat pulpitis via the TNFR2/14-3-3ε-NF-κB pathway, highlighting its potential as a strategy for vital pulp therapy.