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

ABSTRACT 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.